Homeplus, one of the three largest discount chains and a local unit of Britain's Tesco, ended the nine-hour suspension on Wednesday night, its spokeswoman told AFP.
"We resumed displaying and selling US beef at our stores... as the government said there was little possibility that the beef contaminated with mad cow disease had been imported here," Homeplus said in a statement.
US authorities Tuesday reported the country's first case of mad cow disease in six years. They stressed there was no danger that meat from the affected dairy cow would enter the food chain.
Homeplus and Lotte Mart -- another South Korean retail giant -- suspended US beef sales on Wednesday morning.
Lotte Mart will not resume the sales for the time being, its spokesman said, adding it needs more time to monitor the situation.
The Seoul government said Wednesday it would not ban US beef imports but vowed to strengthen inspections.
US beef has been a highly contentious issue in recent years in South Korea.
After barring imports in late 2003 following a mad cow case, Seoul agreed in 2008 to resume most beef imports.
The decision sparked a weeks-long wave of mass street protests. Rallies were ostensibly about the dangers of mad cow disease but took on a general anti-government tone.
They died down after Seoul announced it would restrict imports from older cattle, seen as more susceptible to the disease.
South Korea imported 107,000 tonnes of US beef last year, 37 percent of total imports of the meat, according to the agriculture ministry.