Objective To evaluate inhibitory effect of Chlamydia trachomatis plasmid-encoded protein pORF5 on HeLa cell apoptosis induced by tumor necrosis factor-alpha(TNF-α). Methods The recombinant lentiviral expression vector containing pORF5 gene and helper plasmids were co-transfected into 293T cells to prepare the recombinant lentivirus. Then, the lentivirus particles were collected and concentrated, and used to infect HeLa cells. Flow cytometric screening identified stable pORF5-expressing HeLa (pORF5-HeLa) cells. Meanwhile, the empty plasmid was transfected into HeLa cells to prepare control HeLa cells. The two cell lines were both divided into two subgroups to be treated with 20μg/L TNF-αand fresh culture medium respectively for 6 hours. Then, Hoechst 33258 staining was performed to observe morphological changes of apoptotic cells, flow cytometry to detect cell apoptosis, real-time PCR to measure the mRNA expression of Caspase3, Bax and Bcl-2, and Western blot analysis to determine the protein expression of Bax and Bcl-2. Results After 6-hour treatment with TNF-α, Hoechst 33258 staining showed variable degrees of karyopyknosis and karyorrhexis, and highly-refractive blue apoptotic bodies in the pORF5-HeLa cells and control HeLa cells. The pORF5-HeLa cells and control HeLa cells both showed significantly higher apoptosis rate in the treated subgroup than in the untreated subgroup (pORF5-HeLa cells:35.5%± 4.5%vs. 9.5%± 1.5%, t=13.53, P<0.01;control HeLa cells:63.6%± 5.8%vs. 7.9%± 0.9%, t=32.36, P<0.01). Compared with treated control HeLa cells, treated pORF5-HeLa cells showed significant decreases in mRNA expression of Bax(72.8%)and Caspase 3(84.5%)(t = 35.29, 42.25, respectively, both P < 0.01), as well as in Bax protein expression(t = 17.58,P < 0.01), but significant increases in Bcl-2 mRNA and protein(6.8 times)expression(t = 87.12, 18.93, respectively, both P <0.01). Conclusion pORF5 plasmid protein can inhibit TNF-α-induced HeLa cell apoptosis likely by increasing the expression of anti-apoptotic protein bcl-2 and decreasing the expression of pro-apoptotic proteins Caspase-3 and Bax.