The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis. Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells. The open reading frame (ORF) encoding the CT440 protein from the C. trachomatis serovar D genome was cloned into the prokaryotic expression vector pGEX-6p and expressed as a glutathione-S-transferase (GST) fusion protein in E. coli XL1-Blue. The CT440 fusion protein was used to immunize mice to raise antigen-specific antibody. After verification by Western blot and immunofluorescence assay (IFA), the specific antibody was used to localize the endogenous CT440 protein and to detect its expression pattern in Chlamydia-infected cells. Cytosolic expression of CT440 in HeLa cells was also carried out to evaluate the effect of the CT440 protein on the subsequent chlamydial infection. The results showed that the hypothetical protein CT440 was localized in the C. trachomatis inclusion membrane, and was detectable 12 h after chlamydial infection. Expression of CT440 in the cytoplasm did not inhibit the subsequent chlamydial infection. In summary, we have identified a new inclusion membrane protein that may be an important candidate for understanding C. trachomatis pathogenesis.