In this study we attempted to identify whether Commerson's leaf-nosed bat (Hipposideros commersoni) is the reservoir of Shimoni bat virus (SHIBV), which was isolated from a bat of this species in 2009. An alternative explanation is that the isolation of SHIBV from H. commersoni was a result of spill-over infection from other species, particularly from the Egyptian fruit bats (Rousettus aegyptiacus), which frequently sympatrically roost with H. commersoni and are known as the reservoir of the phylogenetically related Lagos bat virus (LBV). To evaluate these hypotheses, 769 bats of at least 17 species were sampled from 18 locations across Kenya during 2009?2010. Serum samples were subjected to virus neutralization tests against SHIBV and LBV. A limited amount of cross-neutralization between LBV and SHIBV was detected. However, H. commersoni bats demonstrated greater seroprevalence to SHIBV than to LBV, and greater virus-neutralizing titers to SHIBV than to LBV, with a mean difference of 1.16 log(10) (95% confidence intervals [CI]: 0.94-1.40; p<0.001). The opposite pattern was observed for sera of R. aegyptiacus bats, with a mean titer difference of 1.06 log(10) (95% CI: 0.83-1.30; p<0.001). Moreover, the seroprevalence in H. commersoni to SHIBV in the cave where these bats sympatrically roosted with R. aegyptiacus (and where SHIBV was isolated in 2009) was similar to their seroprevalence to SHIBV in a distant cave where no R. aegyptiacus were present (18.9% and 25.0%, respectively). These findings suggest that H. commersoni is the host species of SHIBV. Additional surveillance is needed to better understand the ecology of this virus and the potential risks of infection to humans and other mammalian species.