Matsatsantsa prevailed thanks to a fine 14th minute strike by Sibusiso Khumalo, who went off injured soon after, and an own goal by Urban Warriors defender Thulani Hlatshwayo.
Edwin Gyimah opened the scoring with an early own goal, and was later sent off after receiving his second caution at Lucas Moripe Stadium.
United joined Golden Arrows - who parted ways with coach Manqoba Mngqithi today - and Free State Stars in the next round of the competition.
Sameehg Doutie and Mame Niang were included in the starting 11 for SuperSport United. Doutie, who once played for Ajax Cape Town, was preferred to Bennet Chenene, while Niang kept his spot with Bradley Grobler denied a reunion with Ajax after picking up a knee injury.
Franklin Cale started against his former employers, while there was a debut for striker Tashreeq Morris.
The Urban Warriors went into the encounter looking to exact some revenge following what happened in the first round of the competition last year.
Matsatsantsa beat the Urban Warriors in the Mother City to go through to the quarter-final stage.
Ajax were on course as early as the fourth minute thanks to an own goal from Gyimah. The Ghanaian deflected Lance Davids's attempt from distance past a wrong-footed Ronwen Williams to give the visiting side an early lead.
SuperSport United, though, looked the stronger side and deservedly levelled through Khumalo 10 minutes later. The fullback raced up field before finding the back of the net via the inside of the post to score his first goal in the blue of SuperSport.
Matsatsantsa took the lead in the 27th minute of the encounter thanks to a Hlatshwayo own goal. Niang set up David Mathebula whose effort in turn was guided past a helpless Anssi Jaakkola.
SuperSport controlled the first stanza, keeping hold of possession and frustrating the visitors. The first half ended 2-1 in favour of the hosts.
The Urban Warriors came out more positive after the interval pressing forward in search of the equaliser.
The away side’s task was made a little easier in the 77nd minute after Gyimah received his second yellow card of the game for apparent time wasting.
The Urban Warriors piled on the pressure in the final 10 minutes, going close through second half substitute Bantu Mzwakali, Davids and Cecil Lolo, whose attempt from range scraped the top of the crossbar.
It was not to be for the Urban Warriors, who remain winless this season.