The Englishman's wicket started a chain reaction leaving the hosts at 171-6 in 20 overs and eventually losing the match by three runs.
England batsman Jason became the first ever batsman to be given out obstructing the field in T20 Internationals (T20I) during his side's three-run defeat against South Africa at The Cooper Associates County Ground, Taunton on Friday.
Chasing a target of 175, England were on course of a series-clinching win with 133/2 in the 16th over bowled by Chris Morris when Roy, who was batting superbly on 67, was given out.
Debutant Liam Livingstone dabbed the ball to the backward point region where South African pacer Andile Phehlukwayo collected it and threw at the non-striker's end. Roy, who was at the non-striker's end, was halfway down the pitch when he was sent back by Livingstone.
Phehlukwayo's throw hit Roy on his boot and the tourists, led by Morris started appealing for obstructing the field and umpire Michael Gough immediately signalled a dead ball and went upstairs with a soft signal not out.
The replays showed that the right-handed batsman did change his path and came in way of the throw. The third umpire took a lot of time and in the end gave the decision in South Africa's favour.
The wicket triggered a mini-collapse as the hosts slumped from 133-2 in 15 overs to 171-6 in 20 overs and eventually lost the match by three runs.
England required 12 from the final over but Phehlukwayo, bowling yorker length outside the off stump, restricted the hosts to singles off the first three balls and debutant Liam Livingstone was run out after confusion over a second run.
In the end, England could manage 171 runs for the loss of six wickets.
Morris, the Man of the Match, was pick of the Proteas bowlers as he scalped two crucial wickets giving away just 18 runs in his four overs.
Debutant Tom Curran had earlier taken 3-33 to restrict the AB de Villiers' side to what looked to be an under-par 174-8.
England had crushed the tourists by nine wickets in the first match of the series in Southampton. The third and final game will be played on Sunday in Cardiff.
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 "England, Liam Livingstone, Chris Morris, Jason Roy, South Africa, Andile Phehlukwayo, Michael Gough, Cricket, England v/s South")
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