We investigated the extent to which the observed gender differences in mental rotation ability among the 2,468 freshmen studying engineering at a Midwest public university attributed to the gender bias of a test. The Revised Purdue Spatial Visualization Tests: Visualization of Rotations (Revised PSVT:R) is a spatial test frequently used to measure students' spatial visualization ability in three-dimensional mental rotation in science, technology, engineering, and mathematics fields. With two major approaches for evaluating measurement invariance, we found that five items in the Revised PSVT:R showed a difference in the response pattern by gender, but the impact of these biased items was marginal on the total scores on the scale. Our findings support the equitable use of the Revised PSVT:R by gender for educational research and practices.