Src is a known proto-oncogene and its aberrant activity is involved in a variety of cancers, including ovarian cancer, whereas the regulatory mechanism of Src has not been fully clarified. In this study, we identified tripartite motif-containing (TRIM) 50 as a novel negative regulator of Src protein. Our data showed that TRIM50 directly interacted with SH3 domain of Src via its B-box domain; and TRIM50 reduced Src stability by inducing RING domain-dependent K48-linked poly-ubiquitous modification. We further demonstrated that TRIM50 acted as a tumor suppressor in ovarian cancer cells by its negative regulation of Src protein. In vivo animal model verified that TRIM50 inhibited the xenograft tumor growth of ovarian cancer by suppressing Src protein. Clinical investigation showed that expression of TRIM50 in clinical specimens was inversely correlated with the clinical stages, pathology grades and lymph node metastatic status of the patients, which indicated the involvement of aberrant TRIM50 expression in disease progression. Further analysis verified the negative correlation between TRIM50 and Src expression in clinical specimens. Altogether, we identified TRIM50 as a novel suppressor of Src protein, and demonstrated that TRIM50 inhibited ovarian cancer progression by targeting Src and reducing its activity, which provided a novel therapeutic strategy for Src over-activated cancers by positive regulation of TRIM50.