OBJECTIVE: Ubiquitin-specific protease 4 (USP4) is a deubiquitinating enzyme with key roles in the regulation of TGF-β1 signaling, suggesting its importance in tumorigenesis. However, the underlying mechanisms causing this are not entirely clear. In the present study, we investigated the effect of USP4 on invasion and tumorigenesis of breast cancer cells, and explored its mechanism. MATERIALS AND METHODS: Effects of USP4 overexpression or USP4 silencing by small interfering RNA (USP4 siRNA) on invasion of breast cancer MDA-MB-231 and T47D cells in vitro was detected. Using siRNAs and inhibitors to examine the USP4 signaling pathway. RESULTS: The migration and invasion assays showed that USP4 promotes human breast cancer cell migration and invasion by USP4 overexpression, and knockdown of USP4 by siRNA inhibits human breast cancer cell migration and invasion. Treatment with RLX siRNAs, TGF-β1 siRNAs, Smad2 siRNAs or BB94 (MMPs inhibitor) to USP4-overexpressing breast cancer cells revealed that USP4- induced RLX via TGF-β1 pathway promotes the cell migration and invasion. Further studies demonstrated that USP4-mediated TGF-β1 activation not only enhances the phosphorylation of Smad2 through TGF-β, but also directly upregulate matrix metalloproteinase (MMP)-9-mediated cell migration and invasion of breast cancer cells. CONCLUSIONS: Therapies targeting the USP4 inhibits invasion of breast cancer cells via Relaxin/TGF-β1/Smad2/MMP-9 signal. These results indicate that USP4 is an attractive target for breast cancer therapy.