Defoe started his first England game since 2013 as the Sunderland striker was selected at the tip of the home side's attack, while Michael Keane made his competitive debut for the Three Lions in central defence due to the absences of Chris Smalling and Gary Cahill.
Lithuania, meanwhile, were captained by 25-year-old Poland-based striker Fiodor Cernych, who entered the match as the top scorer in their squad on eight international goals. Nerijus Valskis was also handed a start in the final third despite only having one international goal to his name in 12 previous appearances.
As expected, it was England that dominated the ball in the opening 10 minutes of action, but Lithuania's defensive shape made it difficult for the home side to find any real space in the final third of the field.
Adam Lallana had a half-chance in the 13th minute after a loose ball fell to the Liverpool attacker in a dangerous position, but his effort was deflected and easily gathered by Lithuania goalkeeper Ernestas Setkus.
The pressure was building, however, and England finally made the breakthrough in the 21st minute when Defoe, who had missed an earlier chance after being found by Lallana, converted a low Raheem Sterling cross to justify Southgate's faith in a player that is being tipped to travel to Russia next summer.
Lithuania had a half-chance to respond just before the 30-minute mark when Cernych found space in a dangerous position, but the number 22 sent his low effort wide of Joe Hart's post, before the lively Defoe missed the target with a powerful effort down the other end moments later.
Bertrand and Kyle Walker were not afraid to get forward in the 15-minute spell before the break as England looked to double their lead, but it was Lithuania that had the final chance of the first period when Vykintas Slivka made use of a defensive mistake before beating Hart to a high ball, but John Stones was on hand to clear off the line.
England should have doubled their lead two minutes into the second period when Bertrand picked out Sterling with a cross from the left, but the Manchester City attacker could not convert at the far post.
Defoe was next to come close for England in the 51st minute after breaking into the Lithuania box, but the Sunderland forward's effort was straight at Setkus as one goal continued to separate the two teams in London.
Slivka had Lithuania's first shot on target in the second period as he tested Hart with an effort in the 53rd minute, before Oxlade-Chamberlain forced Setkus into a flying save down the other end moments later.
Marcus Rashford and Vardy replaced Defoe and Sterling on the hour as Southgate looked for more invention in the final third during a period where Lithuania were growing in confidence. Arturas Zulpa was next to try his luck for the visitors in the 63rd minute, but the midfielder's long-range effort was too high.
England doubled their lead in the 66th minute, however, when Vardy slotted into the bottom corner from close range after a clever flick from Lallana had released the Leicester City forward into the Lithuanian box.
Rashford tried his luck from distance 20 minutes from time as the Three Lions looked for more goals, before Dele Alli brought a save from Setkus as the pressure continued to build on the away side's penalty box.
Vardy was the target of two dangerous crosses within the space of 60 seconds 10 minutes from time at Wembley, but strong defending from centre-back Tadas Kijanskas saw the visitors clear their lines.
Vardy had another opportunity moments later after a ball from Rashford had released the former non-league striker, but he lifted his effort over the crossbar under pressure from the onrushing Setkus.
The pressure from England continued into the latter stages as Alli headed a Rashford cross just wide in the 88th minute, but it finished 2-0 on the night as Southgate's side moved five points clear of second-placed Slovenia at the top of Group F.