The Tottenham striker provided head coach Roy Hodgson with a slice of revenge for the Euro 2012 quarter-final defeat against the Italians.
Daniele de Rossi headed Italy in front but Phil Jagielka levelled before half-time and then substitute Defoe struck with 10 minutes left.
After the game England manager Roy Hodgson told ITV: "It gave us a chance to see so many new players - it might have been different if we'd had to use the same players we'd used through the Euros.
"It was great to give the senior players a rest and blood so many new players, or players who didn't get the chance before, and I thought they answered the challenge in the best possible way."
Hodgson signalled out debutant Tom Cleverley for special praise, saying of the Manchester United youngster: "We know he's a good player and he's a very, very important player for Man United.
"Last year they were very unlucky to lose him so early and we've been pleased to see him back in form so early, and he must be pleased to add to that with a good performance for the England first team."
Hodgson even found cause for optimism since the Euro 2012 semi-final disappointment, adding: "There was not a lot different from what we were trying to do in the Euros, but maybe tonight we showed a little bit more composure on the ball and managed to pass it a bit better."
Match-winner Jermain Defoe admitted his second half strike was one of the best of his England career but added: "Even people who have had more England caps than me would say that each time they pull on an England shirt they want to prove something to the manager."
And debutant Cleverley added: "It was a proud moment for me and a big moment in any footballer's life. I managed to get forward at times and hopefully I can impress the manager and get called up again."
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