L. monocytogenes is a widespread facultative intracellular pathogen. The range of natural hosts that supporting L. monocytogenes persistence in the environment has not been fully established yet. In this study, we were interested in the potential of L. monocytogenes to infect cells of bats, which are being increasingly recognized as a reservoir for microorganisms that are pathogenic to humans and domestic animals. A stable epithelial cell line was developed from the kidneys of Pipistrellus nathusii, a small bat widely distributed across Europe. The wild-type L. monocytogenes strain EGDe infected this cell line with an invasion efficiency of 0.0078 ± 0.0009%. Once it entered bat cells, L. monocytogenes doubled within about 70 minutes. When L. monocytogenes lacked either of the major invasion factors, InlA and InlB, invasion efficiency decreased by a factor of 10 and 25 respectively (p < 0.000001). The obtained results suggest that bat epithelial cells are susceptible to L. monocytogenes infection and that L. monocytogenes invasion of bat cells depends on the major invasion factors InlA and InlB. These results constitute the first report on in vitro studies of L. monocytogenes infection in bats. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.