UEFA Europa League: Tottenham 2 Sheriff 1
The striker, who equalled Martin Chivers' record in the home win over the same opponents, scored a penalty to move on to 23 goals in continental competition to earn a place in the club's record books.
Manager Andre Villas-Boas side went into the game knowing victory would see them through to the next stage as they seek their first European trophy since the UEFA Cup triumph in 1984.
Tottenham won the previous encounter between the two sides 2-0 and they had little difficulty in making it a double against the Moldovan outfit.
Erik Lamela gave the hosts the lead with a smart low finish 15 minutes into the second half, the Argentinian scoring his first goal for the club, before Defoe's historic strike from the spot.
Ismail Isa did pull one back for the visitors but it proved inconsequential as Tottenham comfortably held on.
Etienne Capoue made his first appearance since September for Tottenham after recovering from an ankle injury, while Brad Friedel came in for goalkeeper Hugo Lloris, who was rested after being knocked unconscious in the 0-0 draw with Everton last Sunday.
Tottenham looked to dominate proceedings early on, but they struggled to create chances, although Capoue should have done better when he dragged an effort wide after 10 minutes.
Sheriff looked to play on the counter-attack for the most part and they almost took a shock lead as Bulgarian striker Isa's deflected effort smacked against the right-hand post following a quick breakaway.
The hosts continued to dictate play in the closing stages of the half, Defoe failing to connect with Gylfi Sigurdsson's right-wing cross just before the break.
Sheriff defended well throughout, but they had their goalkeeper Vjekoslav Tomic to thank for keeping them on level terms as he produced a stunning save to prevent Christian Eriksen's fierce drive from finding the net.
However, Villas-Boas men did finally break the deadlock on the hour mark as Lamela turned smartly to fire home on the half-volley after Eriksen's pass had deflected into this path.
And victory looked to be all but confirmed seven minutes later, Defore etching his name into the history books with an emphatic penalty following Djbril Paye's clumsy challenge on Lamela.
Sheriff refused to lie down, though, forward Isa reacting quickest to a rebound at the near post after Friedel had saved well from Cadu.
A nervy finish was never on the cards, however, as Tottenham cruised to a deserved win.