Steyn has not recovered from a side strain suffered during a warm-up match against Pakistan on Monday, and skipper AB de Villiers hinted that the strike bowler will be kept out for the first match.
"It's not looking good," de Villiers told reporters before the team's training at the Cardiff Wales stadium.
"It's sort of touch-and-go. It is obviously something we will have to look at. There are a few games coming up, so we would not like to get him out for the rest of the tournament."
Steyn's likely absence spells further trouble for the Proteas, who are already without the veteran duo of Graeme Smith and Jacques Kallis, while the captain himself is vulnerable to injury.
Left-handed batsman Smith was ruled out of the tournament with an ankle injury, while seasoned all-rounder Jacques Kallis opted out to take a voluntary break from cricket.
De Villiers, who has suffered a spate of injuries over the past year, insisted he was fit to play, but the Proteas have called up reserve wicket-keeper Quinton de Kock as a precautionary measure.
"Quinton is here if something happens in a game," said de Villiers, who also keeps wickets besides batting at the top of the order.
"If I get injured tomorrow, we are going to need a wicket-keeper. But I am 100 percent. There is absolutely nothing wrong with me."
The Proteas are looking to win their first major title since their victory in the inaugural ICC knockout tournament in Bangladesh in 1998, an event that later became the Champions Trophy.
India, the reigning World Cup champions and the top-ranked one-day side, flexed their prowess ahead of the eight-nation tournament with two remakable victories in the warm-up games.
Mahendra Singh Dhoni's men first chased down Sri Lanka's 333 with an over to spare, before hammering Australia by a whopping 243 runs as they discovered timely form with bat and ball.
Dinesh Karthik ensured he his place in the playing XI with an unbeaten 106 off 81 balls against Sri Lanka, followed by a superb 146 not out against the Australians.
"I have always believed that Dinesh can bat really well in the middle-order," Dhoni said. "He reads the situation pretty well and he is someone who can go on and play a big innings."
India's squad comprises just three players -- Dhoni, Virat Kohli and Suresh Raina -- who took part in the World Cup final against Sri Lanka in Mumbai two years ago.
India may be one of the pre-tournament favourites, but they have lost five of their last seven internationals against South Africa, including a three-wicket defeat in the World Cup in Nagpur.
Dhoni said the new one-day rules, which allowed just four fielders outside the circle instead of five, meant he faced a tricky choice to find the right combination.
"We may have to go in with five bowlers," he said. "With the new rules, it will be very difficult to play just four bowlers and rely on part-timers."
Pakistan and the West Indies are the other two teams in group B, from which the top two will qualify for the semi-finals.
Group A comprises defending champions Australia, Sri Lanka, New Zealand and hosts England.
The final is scheduled for June 23.
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