Daniele De Rossi's header after a quarter of an hour was cancelled out by Phil Jagielka's first international goal, again via a header from a corner, as the half-hour mark approached.
Gary Cahill had a goal disallowed midway through the second half before Italy had goalkeeper Salvatore Sirigu to thank for averting an own goal from Federico Peluso, but England were not to be denied a small measure of revenge for their quarter-final exit at the hands of the Azzurri 52 days earlier when Defoe rounded off a quick counter-attack with a sweet strike from the edge of the box with 10 minutes remaining.
The win was England's first over Italy since a 2-0 victory at Le Tournoi in 1997.
The match was part of what could be the final round of August international friendlies and the widespread apathy these fixtures are generally met with was shown by the half-full stadium and the teams both managers sent out.
There were several withdrawals and absentees for both sides for reasons of varying validity, but the result was two experimental line-ups who served up a reasonably entertaining match.
England started marginally the better of the two sides, who were playing out a rematch of their Euro 2012 quarter-final, but an Andy Carroll header sent well over the bar was the only reward for their efforts.
At the age of 19 years and 158 days debutant Jack Butland became England's youngest ever goalkeeper, and he showed his potential early on when he reacted quickly to turn a deflected free-kick around the foot of the post.
However, from the subsequent corner De Rossi lost marker Michael Carrick far too easily and guided his header over Butland into the roof of the net.
Italy then started to take control, with Mattia Destro beating the offside trap and deftly taking a floated high ball on his chest, only for Kyle Walker to recover enough ground to put the new Roma striker off at the crucial moment.
Just as Italy had been on the back foot before their goal, so England found their equaliser having withstood a period of pressure.
Frank Lampard, wearing the captain's armband as he did when England beat Spain last November, sent in a 28th-minute corner that Jagielka guided past Sirigu with a well-executed diving header.
Lampard will turn 36 during the finals of 2014 World Cup, but he showed he can still be of value in at least the qualifying stages for Brazil with an assured performance that also featured a strike from range which was beaten away by Sirigu and a free-kick that skimmed the roof of the net.
As half-time approached AC Milan full-back Ignazio Abate fired a strike across the face of goal after a slick one-two with Alessandro Diamanti, but it remained 1-1 at the break.
Instead of the anticipated slew of changes at half-time there were just three substitutions. One of those saw John Ruddy replace Butland for his own England debut, while Defoe came on for Carroll and Peluso replaced Balzaretti for Italy.
Ruddy was soon called into action by the lively Destro, parrying the striker's low shot at the near post. As both managers began to make more changes it was England who began to take hold of the match, with second-half introduction James Milner particularly influential.
Cahill thought he had put England ahead when he volleyed home a corner from the penalty spot, only for the referee to disallow it for Joleon Lescott tussling with his marker.
Soon afterwards Milner whipped in an excellent delivery that caused Peluso to almost beat his own keeper, but Paris Saint-Germain keeper Sirigu managed to show great reflexes and scoop the ball away off the line. Lescott could only guide the rebound onto the bar before a relieved Italy cleared.
Ruddy was then called into action again when debutant Marco Verratti – a less heralded summer recruit for PSG – forced him into tipping a strike over the bar, and from the subsequent corner England scored.
Ryan Bertrand had not been on the pitch for long when his clearance sent Defoe galloping up the pitch on the break. The Tottenham striker created half a yard of space off Abate and his thundering shot from the edge of the was too solid for Sirigu's fingertips to prevent it from flying into the far corner.
Defoe almost got another late on when more great work from Milner led to a low cross into the box, but the ball was diverted wide at the last moment.
The win should give England renewed confidence heading into their opening World Cup qualifying match away to Moldova next month, while Italy will open their 2014 campaign in Bulgaria.