Barriers to animal movement can isolate populations, impacting their genetic diversity, susceptibility to disease, and access to resources. Barriers to movement may be caused by artificial light, which is known to disrupt bird, sea turtle, and bat behavior, but few studies have experimentally investigated the effects of artificial light on movement for a suite of terrestrial vertebrates. Therefore, we studied the effect of ecological light pollution on animal usage of a bridge under-road passage structure. On a weekly basis, sections of the structure were subjected to different light treatments including no light added, followed by a Reference period when lights were off in all the structure sections. Sand track data revealed use by 23 mammals, birds, reptiles and amphibians, nine of which had > 30 tracks for species-level analysis. Columbia black-tailed deer (Odocoileus hemionus columbianus) traversed under unlit bridge sections much less when neighboring sections were lit compared to when none were, suggesting avoidance due to any nearby presence of artificial light. Similarly, deer mouse (Peromyscus maniculatus) and opossum (Didelphis virginiana) track paths were less frequent in the lit sections than the ambient. Crossing was correlated with temporal or spatial factors but not light for three of the other species. These findings suggest that artificial light may be reducing habitat connectivity for some species though not providing a strong barrier for others. Such information is needed to inform mitigation of habitat fragmentation in the face of expanding urbanization.