It happened at Three Mile Water, near Mossley Mill, in Newtownabbey.
A local angling group said hundreds of juvenile and adult trout and salmon were affected.
The Northern Ireland Environment Agency (NIEA) confirmed it had begun an investigation into what caused the fish kill.
In a statement, the NIEA said it was alerted on Tuesday after receiving a water pollution report from Antrim and Newtownabbey Borough Council.
It said a water quality inspector "was able to quickly confirm the presence of a significant number of dead fish".
"From his initial investigation, the river was noted to be running visibly clear with no signs or odours of untreated sewage or agricultural effluents which are often associated with impact on fisheries," a spokesman said.
Water samples have been taken away for testing to confirm if a chemical pollutant caused the fish kill.
Andrew Moore of Three Mile Water Conservation and Angling Association said what happened was a mystery at this stage.
"If you're looking at the river, you wouldn't realise that it's polluted - apart from the dead fish, it's completely clear and there's no smell," he said.
"That's what has us baffled, and I understand it has the Environment Agency baffled too."
An environmental investigation has begun after a "significant" number of fish were killed in a river in County Antrim.