Chlamydia trachomatis is a bimorphic, obligate intracellular parasite that develops entirely within a membrane-bound vacuole. Chlamydiae manipulate eukaryotic hosts by translocating effector proteins into the host cell. Chlamydia genomes encode a type III secretion system (T3SS) which is a likely mechanism for delivery of these effectors. The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early-cycle events. For example, the T3S-dependent chlamydial substrate Translocated actin-recruiting phosphoprotein (Tarp) is present in infected cells as early as 5 minutes post infection, and it is likely that EBs translocate other proteins in a T3S-dependent manner. Evidence is presented herein that the C. trachomatis protein CT694 represents an early-cycle-associated effector protein. CT694 is secreted by the heterologous Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK and the C-terminus of AHNAK2. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. In addition, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibers that correlated with the ability of CT694 to interact with AHNAK. Domain differentiation studies indicated that the C-terminus of CT694 is required to interact with AHNAK and a domain within the N-terminus localizes CT694 to the plasma membrane and triggers aberrant morphological changes in HeLa cells. CT694 function is conserved in other C. trachomatis serovars and C. muridarum, but not by similarly positioned genes of C. pneumoniae or C. caviae. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis, and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development unique to this species.