Midline2 (MID2) is an ubiquitin-conjugating E2 enzyme linked to tumor progression and a novel interacting partner of breast cancer 1, early-onset (BRCA1). However, the role of MID2 in breast cancer remains unknown. This study investigated the expression, prognostic value, and role of MID2 in breast cancer. The expression of MID2 mRNA and protein was significantly upregulated in breast cancer tissue and established cell lines compared with that in normal breast epithelial cells and paired adjacent non-tumor tissue (P<0.001). Immunohistochemical analysis demonstrated that MID2 was overexpressed in 272 of 284 (95.8%) paraffin-embedded, archived breast cancer tissue. Moreover, MID2 expression increased with advanced clinical stage (P<0.001). High MID2 expression was significantly associated with advanced clinical stages and T, N, and M staging (all P<0.05). Univariate and multivariate analyses indicated that high MID2 expression was an independent prognostic factor for poor overall survival in the entire cohort (93.73 vs. 172.1 months; P<0.001, log-rank test) and in subgroups with stages Tis+ I+ II and III+ IV. Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colony formation, and anchorage-independent growth ability assays were conducted. Results showed that siRNA silencing of MID2 expression significantly reduced MCF-7 and MDA-MB-231 cell proliferation in vitro and blocked the growth of MDA-MB-231 cell xenograft tumors in vivo (P<0.05). This study indicated that MID2 may be a novel prognostic marker and interventional target in breast cancer.