Chlamydia trachomatis is a bacterial agent responsible for one of the most prevalent sexually transmitted diseases worldwide. The organism’s unique biological complexity has limited our understanding of the key pathogenic mechanisms, however the HtrA protein has recently by identified as a potential C. trachomatis virulence factor. HtrA is a tightly regulated quality control protein that, upon activation, transforms into functionally active ‘cages’ enabling the degradation or refolding of misfolded substrates, however the specific activation mechanism on an amino acid level has not been defined. To elucidate this mechanism in C.trachomatis HtrA (CtHtrA) a combination of molecular modelling, amino acid mutagenesis, and proteolytic activation and degradation assays were used. Molecular modelling yielded two novel homology models, representing both the active and inactive states of the protein, as well as five specific structural interactions that potentially mediate the allosteric activation of CtHtrA. Point mutations disrupting these interactions revealed that residues R362, R224, and R299 significantly disrupt degradative activity and are integral in stimulating the proper conformation of the active site, while residues K171 and K160 were found to mediate substrate specificity. Cage-forming proteases that are allosterically activated are quite unique and understanding their activation mechanism is critical for unlocking new biological pathways to investigate disease, as well as the generation of new drug targets. This work provides a comprehensive understanding of a novel structural activation cascade in a likely critical pathogenesis protease from Chlamydia trachomatis.