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Two off the last ball, which saw bowler Stuart Broad miss with a shy off the stumps, saw the Dutch home to a total of 163 for six in reply to England's 162 for five.
England who had been 102 for one were made to pay for their failure to build on a superb opening stand by some enterprising Dutch batting led by a fine 49 from Tom de Grooth.
Essex all-rounder Ryan ten Doeschate was 22 not out and Edgar Schiferli five not out.
The Dutch needed seven off the last over. They scrambled a single off the first ball when Stuart Broad missed with an underarm throw at the stumps.
Next ball Broad had another chance but the diving quick knocked the stumps down with his hand rather than the ball to leave the Dutch needing five off four with ten Doeschate on strike.
Broad hen dropped a caught and bowled chance off the next ball which again yielded a single, four runs off three balls the target for the Dutch who scrambled a bye after wicket-keeper James Foster failed to hit the stumps.
That left the target three off two and in turn that became two off one with Schiferli on strike before the minnows got home in a dramatic conclusion to a thrilling match.
While England's Innings hadn't featured a single six, Netherlands opener Darron Reekers managed two off his own bat, pulling Ryan Sidebottom and James Anderson, during his knock of 20.
Meanwhile de Grooth reverse-swept leg-spinner Adil Rashid for four.
Rashid though did have Bas Zuiderent stumped by Foster to leave the Dutch 66 for three in the ninth over.
At the 10 over half-way mark the Netherlands were 91 for three and up with the rate and de Grooth gave England captain Paul Collingwood the same treatment he'd been meeting out to the rest of the attack by pulling the medium-pacer for four through mid-wicket.
After a fumble by Ravi Bopara cost England the chance of a run-out Peter Borren hoisted Collingwood into the Grandstand for six.
De Grooth's impressive 30-ball innings ended with him one short off a fifty when he got a leading edge to a Collingwood slower ball and gave a gentle catch to Robert Key at short cover.
His exit ended a fifty stand with Borren but at 116 for four off 13 overs the Netherlands needed 47 runs off the final seven overs for a famous win.
England missed another run-out chance when Foster fumbled Collingwood's throw with Borren yards short.
Borren though holed out for 30, off just 25 balls, with the Dutch 133 for five in the 16th over as the rain started to fall.
Daan van Bunge then went down the pitch to cream Rashid for four through the covers and the Netherlands's target was down to 21 off three overs.
But when Van Bunge drove Anderson straight to Luke Wright, the Dutch were left with a target of 17 off 12 balls.
The Netherlands earlier staged a gutsy fightback with the ball.
England, who were without the injured Kevin Pietersen (Achilles), had been on a course for a huge total during an opening stand of 102 between Bopara (46) and Luke Wright, who top scored with 71.
But from 102 for none in the 12th over, England lost five wickets for 51 runs as their innings petered out, with ten Doeschate taking two for 35.
Rain forced the opening ceremony to be abandoned and the start delayed by 20 minutes before Netherlands captain Jeroen Smits won the toss and decided to field in damp, murky conditions.
The decision initially backfired as Bopara and Wright put on 102 for the first wicket.
Bopara struck the first ball of the turnament, from Dirk Nannes, for four. Despite a fiery couple of overs from the left-arm fast bowler, the century partnership was reached after 66 balls.
Bopara was soon out, caught at long on by Pieter Seelaar off Ryan ten Doeschate, but Wright, who played a riskier innings, reached his 50 from 36 balls.
However, England's other batsmen struggled to hit boundaries. Owais Shah made 5 before he has caught at deep mid-wicket by Alexei Kervezee off Edgar Schiferli, and Eoin Morgan was out for 6 when he attempted to reverse-sweep Peter Borren and chipped the ball straight to Bas Zuiderent.
Wright was eventually caught by Borren off Ten Doeschate, and captain Paul Collingwood made 11 when Seelaar drew him into a rash shot that was caught by Schiferli.
England conclude their Group B programme against Pakistan at the Oval on Sunday while the Netherlands face Pakistan at Lord's on Tuesday.
Score card
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Netherlands.
| Batsman | Status | R | B | 4's | 6's | SR | Mat | Runs | Ave |
| AN Kervezee | c Stuart Broad b James Anderson | 1 | 4 | 0 | 0 | 25 | 1 | 1 | 1 |
| DJ Reekers | c Owais Shah b Stuart Broad | 20 | 13 | 1 | 2 | 153.85 | 5 | 51 | 17 |
| B Zuiderent | st. James Foster b Adil Rashid | 12 | 16 | 1 | 0 | 75 | 1 | 12 | 0 |
| Tom Nico de Grooth | c Robert Key b Paul David Collingwood | 49 | 30 | 6 | 1 | 163.33 | 5 | 92 | 46 |
| Peter Borren | c Owais Shah b James Anderson | 30 | 25 | 1 | 1 | 120 | 5 | 84 | 42 |
| RN ten Doeschate | not out | 22 | 17 | 1 | 0 | 129.41 | 4 | 106 | 53 |
| DLS van Bunge | c Luke Wright b James Anderson | 8 | 8 | 1 | 0 | 100 | 5 | 34 | 8.5 |
| E Schiferli | not out | 5 | 7 | 0 | 0 | 71.43 | 5 | 5 | 2.5 |
| J Smits (W & C) | |||||||||
| Dirk Nannes | |||||||||
| Pieter Seelaar | |||||||||
| Target: 163 runs from 20 overs | Total:(163/6 in 20.0 overs) | ||||||||
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