Former West Ham and Tottenham forward Defoe has been in prolific form for Sunderland this season, despite David Moyes' side languishing at the bottom of the Premier League, scoring 14 times in 27 top-flight outings.
The last of his 55 international appearances came as a substitute in a November 2013 friendly loss to Chile at Wembley.
Defoe made his debut for the Three Lions in March 2004 versus Sweden and has 19 goals at international level.
Thanks to everyone for your messages! Playing for @England has been my dream since day one so to be back in the squad is so special for me.— Jermain Defoe (@IAmJermainDefoe) March 16, 2017
It's also special for my family and I feel I deserve this because I've always dedicated myself to the game. I'm really looking forward to...— Jermain Defoe (@IAmJermainDefoe) March 16, 2017
Meeting up with the squad and seeing all the boys. This time around it'll be strange being one of the older lads, but I can't wait! BUZZING!— Jermain Defoe (@IAmJermainDefoe) March 16, 2017
Manchester United teenager Marcus Rashford and Leicester City's Jamie Vardy join Defoe in the striking department and there are surprises elsewhere in Southgate's 26-man squad.
Walcott, who's been a constant in the Three Lions set-up in recent years, will watch from the sidelines this international break.
Southampton duo James Ward-Prowse and Nathan Redmond join West Ham's Michail Antonio and Burnley defender Michael Keane in aiming to win their maiden caps, while West Brom midfielder Jake Livermore comes in after a longer international exile than Defoe – his solitary England appearance being from the bench in a 2-1 win against Italy in August 2012.
Luke Shaw is included despite the left-back only having played twice for Manchester United since November 30.
England head to Dortmund for a friendly against world champions Germany next Wednesday, before looking to solidify their position at the top of Group F in the European qualifiers for Russia 2018 when they host Lithuania at Wembley on March 26.
England squad in full:
Goalkeepers: Fraser Forster (Southampton), Joe Hart (Torino), Tom Heaton (Burnley)
Defenders: Ryan Bertrand (Southampton), Gary Cahill (Chelsea), Nathaniel Clyne (Liverpool), Phil Jones (Manchester United), Michael Keane (Burnley), Luke Shaw (Manchester United), Chris Smalling (Manchester United), John Stones (Manchester City), Kyle Walker (Tottenham)
Midfielders: Dele Alli (Tottenham), Michail Antonio (West Ham), Ross Barkley (Everton), Eric Dier (Tottenham), Adam Lallana (Liverpool), Jake Livermore (West Brom), Alex Oxlade-Chamberlain (Arsenal), Nathan Redmond (Southampton), Raheem Sterling (Manchester City), James Ward-Prowse (Southampton)
Strikers: Jermain Defoe (Sunderland), Marcus Rashford (Manchester United), Jamie Vardy (Leicester City).