The accident happened on State Highway 29 in Karapiro, 10km southeast of Cambridge, about 10.30pm, police northern communications Inspector Ian Booker said.
One man died at the scene, and the second was taken to Waikato Hospital in a serious condition.
It was not known how the cow, which was killed in the crash, came to be on the road, Mr Booker said.
A section of the road between Hinuera and the eastern end of Lake Karapiro was closed as serious crash investigators worked at the scene.
