Chlamydiae are bacterial pathogens which have evolved efficient strategies to enter, replicate, and survive inside host epithelial cells, resulting in acute and chronic diseases in humans and other animals. Several candidate molecules in the host receptor complex have been identified, but the precise mechanisms of infection have not been elucidated. Epithelial membrane protein-2 (EMP2), a 4-transmembrane protein, is highly expressed in epithelial cells in sites of chlamydial infections. Here we show that infectivity of the Chlamydia muridarum (MoPn) is associated with host cellular expression of EMP2 in multiple cell lines. Recombinant knockdown of EMP2 impairs infectivity, whereas infectivity is augmented in cells recombinantly modified to over-express EMP2. An epithelial cell line without native expression of EMP2 is relatively resistant to MoPn infection, whereas infectivity is markedly increased by recombinant expression of EMP2 in that cell line. Blockade of surface EMP2 using a specific anti-EMP2 antibody significantly reduces chlamydial infection efficiency. In addition, MoPn infectivity as measured in the EMP2 overexpressing cell line is not heparin-dependent, suggesting a possible role for EMP2 in the non-reversible phase of early infection. These findings identify EMP2 as a candidate host protein involved in infection of C. muridarum (MoPn).