The National Union of Metalworkers of South Africa (Numsa) announced the break at a special congress of members.
"Numsa as an organisation will neither endorse nor support the ANC or any other party in 2014," said leader Irvin Jim, who also called on President Jacob Zuma to resign.
The announcement could spell trouble for South Africa's ruling "tripartite" alliance, which has helped the ANC coast to victory in every elections since the end of white rule in 1994.
The ANC has entered each election with vital logistic and political support from the trade union umbrella group Cosatu - of which Numsa is the biggest member - and the Communist Party.
With more than 300,000 members, Numsa is an influential source of votes, cash and grass-roots organisation. Many members of Numsa are also members of the ANC.
Numsa also called for Cosatu to withdraw its support.
"The congress called on President Jacob Zuma to resign with immediate effect because of his administration's pursuit of neo-liberal policies... steeped in corruption, patronage and nepotism," Jim said.
He also floated the idea of establishing a new socialist party.
"The time for looking for an alternative has arrived," he told hundreds of cheering members at the end of a four-day congress.
The union also called for a parallel investigation into the police killing of 34 miners at Marikana, which is already being looked into by a government-appointed commission.