Triple negative breast cancer (TNBC) is one of the most common malignant tumors in women and the tripartite motif 66 (TRIM66) is closely associated with the behaviors of wide variety of cancer. Thus, in this study, we aimed to explore the effect of TRIM66 in MDA-MB-468 TNBC cell line. Western blot and RT-PCR assays were used to detect the expression level of TRIM66 in human normal mammary cells and human breast cancer cell lines. We silenced its expression in MDA-MB-468 cells by transient siRNA transfection to ascertain the function of TRIM66 in MDA-MB-468 cells. CCK8 and clonogenicity assays were used to evaluate the ability of cell proliferation. Wound healing and Transwell were used to detect cell invasion and migration. TUNEL was applied to detect the apoptosis level. Western blotting was used to detect the expression of related proteins. TRIM66 was observed markedly elevated in breast cancer cell line. Knockdown of TRIM66 inhibited the expression of EGFR, P-Jak2, P-STAT3, JAK2, and STAT3 in MDA-MB-468 cells through regulating the epidermal growth factor receptor (EGFR) signaling. Knockdown of TRIM66 suppresses proliferation, invasion and migration of MDA-MB-468 cell line and promotes apoptosis in these cells through EGFR signaling.