Without a win in their first three games of the season, Tottenham took the lead after 18 minutes when Gylfi Sigurdssdon and Aaron Lennon combined to set up Defoe for a cool finish.
Gareth Bale made it 2-0 after 71 minutes with a scuffed shot from Kyle Walker's cut-back and Defoe continued his hot streak with a superb solo goal, running from inside his own half to clip a shot across Reading goalkeeper Alex McCarthy.
Reading scored a consolation in the 90th minute when Hal Robson-Kanu hooked the ball in at the far post.
Villas-Boas, who failed to complete a season at Chelsea after being sacked in March, had come under pressure after replacing Harry Redknapp at Tottenham but victory lifted his side to 10th in the table with five points.
Promoted Reading are still searching for their first win since returning to the top flight.
Defoe, who spent much of last season as a substitute under Redknapp, has enjoyed a bright start to the campaign, bagging three Premier League goals and one for England in the World Cup qualifier against Moldova.
Starting as a lone striker, he was a constant menace to Reading's defenders and guided a shot inside the post to put the visitors in front.
Tottenham should have added to their lead as Reading were guilty of giving the ball away in dangerous areas, Sigurdsson having one close-range effort cleared off the line after Defoe had robbed McCarthy.
Reading enjoyed a little more possession after the break but rarely threatened Spurs keeper Brad Friedel, who kept his place in the starting team ahead of France international Hugo Lloris.
Wales winger Bale scored his first league goal since January to give Tottenham breathing space with 20 minutes to go before Defoe put the result beyond doubt.
"It was a well-deserved win. Everyone performed to their top level and it is a win that will lift us up for the season," Villas-Boas told Sky Sports.
"We created lots of chances and the second goal gave us the stability. It was a good solid performance from everybody. The most important thing today was the motivation of the players to change the negative results at home," the Portuguese added.
"Hopefully we can now build for the season ahead, it takes away the anxiety and gives us a good relaxed week ahead."
Chelsea lead the table after four matches with 10 points following their 0-0 draw at local rivals Queens Park Rangers on Saturday.
Manchester United crushed Wigan Athletic 4-0 to go second on nine points with Arsenal, 6-1 winners over Southampton, and champions Manchester City, who drew 1-1 at Stoke City, on eight.
Everton host Newcastle United on Monday.
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