Objective To investigate the effect of Mesothelin on proliferation, apoptosis, migration and invasion of human triple-negative breast cancer cells (TNBC). Methods Immunohistochemical staining was used to detect the expression of Mesothelin in TNBC tissue, paracancer tissue and breast cancer cell lines. TNBC cell line MD-MB-231 cells were transfected with the overexpressed Mesothelin lentivirus plasmid (the mesothelin group) and the control Scramble plasmid (the Scramble group). MD-MB-231 cells with no infected lentivirus were used as the control group. Flow cytometry was used to detect the efficiency of virus infection. The expression of Mesothelin was detected by qPCR, flow cytometry and Western blot assay. Cell proliferation was detected by CCK-8. Annexin V-APC/PI was used to detect apoptosis. The expression of anti-B lymphoblastoma-2 (Bcl-2), anti-Bcl-2 associated X protein (Bax), E-cadherin, N-cadherin and Vimentin were detected by Western blot assay. Cell migration ability was detected by scratch test. The invasive ability of cells was detected by Transwell assay. Results The expression of Mesothelin was significantly higher in TNBC tumor tissue than that in paracancer tissue (P<0.05), and Mesothelin was expressed in breast cancer cell line. Flow cytometry showed that the efficiency of lentivirus infection was more than 90%. Compared with the Scramble group, the expression level of Mesothelin mRNA was increased in the Mesothelin group, and the cell proliferation rate was increased, while the cell apoptosis rate was decreased. The expression of Bax and E-cadherin protein decreased, the expression of Bcl-2, N-cadherin and Vimentin protein increased, and the migration and invasion ability of cells were significantly enhanced (P<0.05). Conclusion Mesothelin can promote the proliferation, inhibit the apoptosis and increase the ability of migration and invasion of human triple-negative breast cancer MDA-MB-231 cells. [ABSTRACT FROM AUTHOR]