In a statement, the party compared the new rules to government policies in apartheid South Africa.
The regulations, which were announced by athletics' governing body the IAAF Thursday, will limit the testosterone levels of female athletes competing between 400m and a mile.
They are aimed at athletes with Differences of Sex Development and are widely reported to impact South Africa's double Olympic champion Caster Semenya.
"The African National Congress (ANC) notes with great concern the International Association of Athletes Federation's (IAAF) new regulations on female testosterone," read a statement.
"These new regulations infringe on the human rights of athletes, targeting mainly those in East Europe, Asia and the African continent.
"The racial undertones of this cannot go unnoticed. The regulations are a painful reminder of our past where an unjust government specifically legislated laws for certain activists in society to stifle their fight against an unjust system."
From 1948 to 1991, South Africa's minority white ruling group imposed a system of racial segregation on the country. Under the system only white South Africans were allowed to vote.
The ANC said it wanted the South African government to challenge the new regulation, which comes into effect in November, at the Court of Arbitration for Sport (CAS).
"We call on government to challenge this grossly unfair, unjust and blatant racist attempt by the IAAF to the Court of Arbitration for Sport (CAS) and have these regulations set aside," it said.
"The ANC will stand with Caster Semenya in yet another attempt by international sport bodies to exlude and discriminate against her."
The IAAF has yet to respond to CNN's request for comment.
'Ensuring a level playing field'
Semenya, who carried the flag for South Africa at the Commonwealth Games opening ceremony, is hyperandrogenous -- meaning she has elevated testosterone levels. The condition has dogged her athletics career ever since she won the world 800m title as an 18-year-old in 2009.
On Thursday, the double Olympic 800m champion tweeted: "I'm 97% sure you don't like me, but I'm 100% sure I don't care."
The new regulations, unanimously approved by the IAAF, require DSD athletes to reduce their blood testosterone level to below 5nmol/L for a continuous period of at least six months, and maintain those levels continuously for the rest of their athletic career, if they are to compete internationally.
Females who do not abide by the rules will still be allowed to compete in non-international competitions or against men.
Many feel the ruling will unfairly target Semenya, who was forced to undergo gender tests when she emerged onto the world stage as a teenager at the 2009 World Championships, winning the 800m in 1:55.45.
But in a statement, IAAF President Sebastian Coe said Thursday that the federation had a "responsibility to ensure a level playing field."
"The revised rules are not about cheating, no athlete with a DSD has cheated, they are about leveling the playing field to ensure fair and meaningful competition in the sport of athletics where success is determined by talent, dedication and hard work rather than other contributing factors," the Englishman added.
The governing body introduced a testosterone limit in 2011 but in July 2015 Indian sprinter Dutee Chand won a landmark case which allowed inter-sex athletes to run without testosterone-suppressing medication.
CAS suspended the IAAF's ru