"Mr Santana is no longer coach of the senior national team," South African Football Association (SAFA) chief executive Raymond Hack told Reuters.
The departure of the 60-year-old Brazilian came after a dismal run of eight defeats in nine games including friendly defeats by Norway and Iceland last week.
The SAFA said the decision was made after a meeting with Santana on Monday.
"It was unanimously agreed Mr Santana would with immediate effect vacate his current position," said a brief statement.
New SAFA president Kirsten Nematandani had promised to dismiss the coach as part of his election manifesto last month.
Santana was the 15th coach employed by South Africa in as many years.
In 2002 the country also fired Carlos Queiroz three months before the World Cup finals.
Clive Barker was also dismissed six months before South Africa's maiden World Cup finals appearance in France in 1998.
PARREIRA MAY RETURN
The removal of Santana opens the door for a possible return by his compatriot Carlos Alberto Parreira, who resigned in April 2008 when his wife fell ill.
Parreira told South African television last week he would be interested in the job.
Santana was recommended by Parreira when his fellow Brazilian resigned.
The SAFA, who will discuss the vacant position at a meeting on Friday, have put assistant coaches Jairo Leal and Pitso Mosimane in temporary charge.
The team continue their World Cup preparations next month with home friendlies against Japan and Jamaica.
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