Chlamydia trachomatis and C. muridarum, human and mouse pathogens, respectively, share more than 99% of open reading frames (ORFs) but differ in a cytotoxin locus. Presence or absence of cytotoxin gene(s) in these strains correlates with their ability to grow in IFN-γ treated mouse cells. Growth of toxin-positive C. muridarum is not affected in IFN-γ treated cells, whereas growth of toxin-negative C. trachomatis is inhibited. We previously reported that this difference in IFN-γ sensitivity is important to the in vivo infection tropism of these pathogens. Here we describe a phenotypic rescue assay that utilizes C. muridarum gamma irradiated killed elementary bodies (iEB) to rescue C. trachomatis infectivity in IFN-γ treated mouse cells. Rescue by iEB was temporal, maximal early post infection, directly related to multiplicity of iEB infection, and was independent of de novo chlamydial transcription. Lastly, C. muridarum iEB vacuoles and C. trachomatis inclusions were not fusogenic, suggesting the factor(s) responsible for rescue was secreted or exposed to the cytosol where it inactivated IFN-γ induced effectors. Chlamydial phenotypic rescue may have broader utility for the study of other EB associated virulence factors that function early in the interaction of chlamydiae with host cells.