Chlamydia trachomatis is an obligate intracellular pathogen of significant public health importance, being the leading cause of preventable blindness and bacterial sexually-transmitted infections worldwide. Ocular infection (trachoma) is a neglected tropical disease, with Australia being the only developed country to still have a significant trachoma burden, endemic in remote and Indigenous communities. In addition, urogenital infections can cause serious complications including pelvic inflammatory disease and infertility. Conventionally treated with antibiotics, treatment failure is estimated to occur in 6-10% of cases despite no evidence of genetic antibiotic resistance in clinical patient isolates. The mechanisms of treatment failure, persistence and pathogenesis in chlamydia are still largely unknown, with genetic tools available for studying Chlamydia being limited. The dual chaperone-protease HtrA is conserved in most bacterial species and is involved in the pathophysiology of several pathogens; however, the function of C. trachomatis HtrA (CtHtrA) is unclear. Our group recently acquired three mutant C. trachomatis isolates which were selected for resistance to an inhibitor of CtHtrA. Whole genome sequencing of the mutants revealed that all isolates had acquired single-nucleotide variations in genes regulating fatty acid synthesis and recycling pathways, with one recently discovered to serve a pivotal role in membrane phospholipid biogenesis. It is therefore our hypothesis that CtHtrA may play a role in chlamydial membrane composition and stability. We aimed to investigate and characterise the phenotype of these mutants to further develop an understanding of CtHtrA function and the impact of its inhibition. Label-free quantitative proteomic analysis revealed significant fold changes in transcription and translation factors, stress response pathways, type III secretion effectors and outer membrane proteins, with an increased abundance of the protein groups in the mutant isolate versus wild-type. These proteomic results could suggest that HtrA has a central role in regulating C. trachomatis membrane composition and hence the organism’s pathophysiology.