Hook is one of four new starters in a team which also shows three positional changes from the team which took the field against the Springboks in Bloemfontein last weekend.
The new-look centre pairing of Roberts and Tom Shanklin is one of three new combinations with coach Gatland also making changes in the front and back rows of the scrum.
Up front the Ospreys hooker Richard Hibbard takes over the hooker berth from injured Matthew Rees with Gethin Jenkins staying at loosehead and Newport Gwent Dragons' Rhys Thomas coming in at tighthead instead of Adam Jones.
The second row of Ian Gough and Alun-Wyn Jones is unchanged but all three of the back row will be wearing new numbers this week.
Captain Ryan Jones moves to number six to allow Gareth Delve of Gloucester Rugby to come in at number eight and Jonathan Thomas to earn a start at openside flanker instead of Dafydd Jones who drops to the bench.
The replacement hooker jersey is worn by T Rhys Thomas who joined the squad last week as a replacement for the injured Rees. Backs cover on the bench includes scrum half Warren Fury, Andrew Bishop and Blues Wing Tom James.
Head Coach Warren Gatland said: "I am satisfied that we have selected a team which accurately reflects all the hard work in training and the match analysis we have carried out this week.
"We know where we were left wanting in the first Test and we have made selections aimed at improving those areas on Saturday.
"We know what James Hook can do and we believe we need him on the pitch from the start as he is too good a footballer not to be used. He has gone well at full back in training during some pretty intense sessions and he will be able to play a big part in the game from 15.
"Knowing how physical the South African midfield were last week, Jamie is the biggest back we had on the field so he will give us more "go forward" in the centre position.
"All three back row forwards we have chosen have vast experience in the positions they will play on Saturday and we believe it will give us a physical combination capable of improving our performance at the breakdown where we need to improve.
"Some of the combinations in this selection are new but we are very well aware of the capabilities of the boys we have chosen and we know what options they offer us.
"This week we have had to build from the disappointment of a bad defeat but our preparation over the last few days has been great and confidence is building again.
"We want to win the respect of our opponents and I am confident we have picked a side capable of earning that respect if they play to their full potential.
"We are playing the World Champions on their home ground but this Welsh side is learning and developing with every game.
"As a coaching team and a squad this week we have learnt a lot of lessons about how to build from our first defeat together and that will make us stronger in the long run."
Wales: James Hook; Mark Jones, Tom Shanklin, Jamie Roberts, Shane Williams; Stephen Jones, Gareth Cooper; Gethin Jenkins, Richard Hibbard, Rhys Thomas, Ian Gough, Alun-Wyn Jones, Ryan Jones (captain), Jonathan Thomas, Gareth Delve.
Replacements: T Rhys Thomas, Duncan Jones, Ian Evans, Dafydd Jones, Warren Fury, Andrew Bishop, Tom James.
WALES v SOUTH AFRICA - 2nd TEST
Tom SHANKLIN (CARDIFF BLUES - Cap No 993)
DoB: 24.11.79. 6'2" / 1.88m. 15st 9lbs / 99kgs
International record: 53 caps (W 25, L 27, D 1; 90 pts - 18T)
Jamie ROBERTS (CARDIFF BLUES - 1,057)
DoB: 08.11.86. 6'4" / 1.93m. 16st 10lbs / 107kgs
International record: 2 caps (W 1, L1; 5 pts - 1T)
Gethin JENKINS (CARDIFF BLUES - Cap No 1,006)
DoB: 17.11.80. 6'2" / 1.88m. 18st 9lbs / 118kgs
International record: 59 caps (W 25, L 32, D 2; 15 pts - 3T)
T Rhys THOMAS (CARDIFF BLUES - Cap No 1,035)
DoB: 23.04.82. 6'0" / 1.83m. 16st 8lbs / 105kgs
International record: 26 caps (W 10, L 15, D 1; 5 pts - 1T)
Tom JAMES (CARDIFF BLUES - Cap No 1,055)
DoB: 17.04.87. 6'2" / 1.88m. 15st 1lb / 96kgs
International record: 2 caps (L 2)