The 34-year-old has not featured for his country since November 2013 when he played against Chile.
Defoe, who has 55 England caps, has been in sensational form once again this season despite the Black Cats struggling at the bottom of the Premier League table.
The former West Ham man has scored 14 goals in 27 league games and will be hoping to make an impact later this month.
Southgate has also called up uncapped Southampton duo Nathan Redmond and James Ward-Prowse while Manchester United's Marcus Rashford is also named.
West Brom's Jake Livermore, Burnley's Michael Keane and West Ham's Michail Antonio are also included as England prepare to move closer to the 2018 World Cup by beating Lithuania on March 26.
Southgate told the official England website: "You can have the young players who are hungry and the old players who are hungry as well. I think we can't just look at young players all the time. I think we have to get results now and also plan for the future."
Tottenham forward Harry Kane misses out with an ankle injury while captain Wayne Rooney does not feature because of a leg injury.