Chlamydia trachomatis infection targets the mucosal epithelium, where squamous and columnar epithelia can be found. Research on Chlamydia trachomatis-epithelia interaction has predominantly focused on columnar epithelia, with very little known on how Chlamydia trachomatis interacts with the squamous epithelium. The stratification and differentiation processes found in the squamous epithelium might influence chlamydial growth and infection dissemination. For this reason, 3D stratified squamous epithelial cultures were adapted to mimic the stratified squamous epithelium, and chlamydial infection was characterized. Chlamydia trachomatis infection in monolayers and 3D cultures were monitored by immunofluorescence and transmission electron microscopy to characterize inclusion growth and chlamydial interconversion between elementary and reticulate body. We observed that the stratified epithelium varied in susceptibility to Chlamydia trachomatis infection. The undifferentiated basal cells were susceptible to infection, while the terminally differentiated upper layers were resistant. If given access to the basal layer Chlamydia trachomatis is able to disseminate and disrupt the epithelial. This disruption have clinical relevance, such as facilitating secondary infection by other STIs. The use of a punch biopsy in 3D cultures revealed that infected samples were unable to close the wound as efficiently as the mock-infected sample. A simplified 2D wound healing assay confirmed these observations. Additionally, this correlated with a reorganization of hemidesmosomes in Chlamydia trachomatis-infected cells but, most importantly, in bystander uninfected cells within the infected sample. The lack of motility and the hemidesmosomes reorganization was shown to be dependent on myosin II contractility and the chlamydial protein CTL0480. This chlamydial protein recruits MYPT1 to the inclusion membrane, which could potentially prevent the cell from controlling the actomyosin tension. In summary, this is the first study to use a 3D stratified epithelial to determine how Chlamydia interacts with this physiologically relevant tissue. Most importantly, this work demonstrates that Chlamydia trachomatis is able to alter the organization of hemidesmosomes which has never been reported for any other bacterial pathogen.