Coalition-backed Iraqi forces in mid-January took control of the sprawling campus of the university, which IS had used as a headquarters during its two-and-a-half-year rule of the city.
Among the many booby-trapped buildings, Iraqi forces found chemicals inside the university, which was heavily damaged in the fighting.
The Iraqis have now run tests on the chemicals and these proved positive for sulphur mustard, Pentagon spokesman Navy Captain Jeff Davis said.
Mosul university was "central to the ISIS chemical weapons program," Davis said, using an IS acronym.
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Davis said he was not aware of anyone having been killed by the chemicals.
"It was used primarily as an irritant and something to scare people," he said.
Iraqi forces began the offensive to retake Mosul in October and have made major advances, though it has been a brutal fight with high casualties.
Most of the east side of the city is in Iraqi hands, though countless booby traps and small IS cells remain.
Meanwhile, the jihadists still control the west of the city.