Objective: To investigate the expression of tetraspanins-29 (Tspan29) in breast cancer tissues and cell lines and to explore the effect of Tspan29 knockdown on proliferation, invasion, migration and epithelial-mesenchymal transition (EMT) of breast cancer MCF-7 and MDA-MB-231 cells. Methods:Atotal of20pairsofbreast cancer tissues and corresponding para-cancerous tissues resected in Minhang Branch of Cancer HospitalAffiliated to Fudan University from June 2017 to February 2018 were collected for this study; in addition, breast cancer celllinesMCF-7,MDA-MB-231andhumanbreastepithelialMDA-kb2cellswerealsocollected.ThemRNAand protein expressions of Tspan29 in above mentioned tissues and cell lines were detected by Real-time quantitative (qPCR) and Western blotting. The expression of Tspan29 in MCF-7 and MDA-MB-231 cells was interfered by siRNA. qPCR was used to detect the mRNA and protein expressions of Tspan29. PCR microarray was used to examine the expressions of EMT-related genes in MCF-7 cells. CCK-8 assayandTranswellwereusedtodetectcellproliferation, migration and invasion of MCF-7 and MDA-MB-231 cells. Results: The mRNA and protein expressions of Tspan29 in breast cancer tissues were significantly higher than that in para-cancerous tissues (all P<0.01); and the mRNA and protein expressions of Tspan29 in MCF-7 and MDA-MB-231 cells were significantly higher than that in MDA-kb2 cells (P<0.01). After being interfered with siTspan29, the mRNA and protein expressions of Tspan29 were significantly down-regulated in MCF-7 cells (all P<0.05); the proliferation, invasion and migration of MCF-7 and MDA-MB-231 cells were significantly inhibited (all P<0.05); and among the EMT-related genes, two were significantly up-regulated while 7 were down-regulated. Conclusion: Tspan29 is significantly up-regulated in breast cancer tissues and cell lines, and knockdown of Tspan29 significantly inhibits the proliferation, invasion and migration of breast cancer cells. ··