The Grand Slam centre has been struggling with stiffness in his achilles all week and has had to withdraw from the squad.
Wales coach Warren Gatland has promoted Blues centre Tom Shanklin from the bench to give the centre his 55th cap while Ospreys midfielder Andrew Bishop moves on to the bench.
Henson’s absence sees Jamie Roberts switch to inside centre to allow Shanklin to take his preferred position at outside centre.
“We wanted to give Gavin the best opportunity to be able to play against South Africa, but after sitting out today’s final session at the Millennium Stadium he told us he wasn’t going to be right for the game,” said Wales assistant coach, Rob Howley.
“We’re quite comfortable with leaving it to this stage or indeed until tomorrow morning as we have Tom Shanklin available to slot into midfield and Jamie Roberts is more than capable of moving into inside centre.
“Gavin has taken part in training sessions on Tuesday and Thursday but has also been rested for some of them including today’s captain’s run. We have had ample opportunity to run the Shanklin and Roberts combination.”
