Aims: The aim of this study was to investigate the invasion and intracellular survival of different Hafnia alvei strains in HeLa cells. Methods and Results: We performed different experiments on the bacterial invasion of different strains of H. alvei into the HeLa cell line using gentamicin protection assays and immunofluorescence. We also report the time course of cell internalization and the effects of inhibitors on the invasion of H. alvei. Levels of invasion varied depending on the conditions (strain, time and inoculum size) used. Conclusions: This study revealed that H. alvei strains were able to enter and persist in a human epithelial cell line. Significance and Impact of the Study: Our in vitro findings highlight the possibility that some H. alvei strains may exploit nonprofessional phagocytes or nonphagocytic cells to spread in vivo, which may be important for the persistence and establishment of an asymptomatic carrier state.