For Chlamydia, an intracellular pathogen of humans, host cell invasion is obligatory for survival, growth and pathogenesis. At the molecular level, little is known about the binding and entry of Chlamydia into the mammalian host cell. Chlamydia are genetically intractable therefore experimental approaches targeting the host are often necessary. CHO6 is a mutagenized cell line resistant to attachment and infection by Chlamydia. In this study, CHO6 was shown using proteomic methods to have a defect in processing of the leader sequence for protein disulfide isomerase (PDI). Complementation by expression of full-length PDI restored C. trachomatis binding and infectivity in the CHO6 mutant cell line. The cell line was also resistant to diphtheria toxin and required complemented cell-surface PDI for toxin entry. These data demonstrate that native PDI at the cell surface is required for effective chlamydial attachment and infectivity.