BACKGROUND: Chlamydia trachomatis is responsible for a widespread sexually transmitted infection. In men, it is associated with a wide clinical spectrum causing infertility. Furthermore, C. trachomatis serovar E infection decreases motility and increases the number of non-viable sperm. No other effects of C. trachomatis have been reported on sperm despite the crucial role of DNA integrity for sperm function. The aim of this study was to investigate the effects of C. trachomatis on sperm apoptosis. METHODS: Sperm from eight normozoospermic men were incubated with increasing concentrations of C. trachomatis serovar E elementary bodies (EB) for 6 and 24 h. Sperm were then collected to evaluate phosphatidylserine (PS) membrane translocation and DNA fragmentation by Annexin V–propidium iodide staining, TUNEL assay and flow cytometry. RESULTS: After 6 h of incubation, C. trachomatis had no effect on the percentage of sperm showing PS externalization. However, a significant effect on this parameter was observed after 24 h. C. trachomatis also significantly increased the number of sperm with DNA fragmentation both after 6 and 24 h of incubation. CONCLUSIONS: C. trachomatis causes sperm PS externalization and DNA fragmentation. These effects may explain the negative direct impact of C. trachomatis infection on sperm fertilizing ability.