The aim of this study was to investigate the expression of mediator complex subunit 27 (MED27) in breast cancer (BC) and explore its effects on the proliferation and apoptosis of BC cells. The expression of MED27 in 60 BC tissues and para-cancer tissues was detected. Based on the significantly high expression level of MED27 in tumors, the tumor samples were divided into high-expression group and low-expression group according to the median standard, with 30 samples in each group. Then, the association between MED27 expression and clinicopathological features of patients was analyzed. The correlation between MED27 expression and survival time of patients was estimated using the Kaplan-Meier method. Next, the expression level of MED27 in cells was also measured using qRT-PCR assay. In vitro study, si-MED27 was designed to interfere with the expression of MED27 in MDA-MB-231 cells. To further explore the mechanism of MED27 in BC, the expression level of SP1 in cells was examined after different treatments. In quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay, MED27 was found to be highly expressed in both BC tissues and cells. Then, the relationship between MED27 expression and clinical pathological data was statistically analyzed, and it was found that MED27 expression was correlated with tumor size and grade. In the 60-month follow-up and Kaplan-Meier analysis, patients with high expression of MED27 had a poor prognosis. In in vitro study, MED27 expression in cells was down-regulated by transfection with si-MED27. Western blot (WB) analysis suggested that si-MED27 could effectively reduce the protein expression level of MED27 in cells, and the specificity protein 1 (Sp1) expression was also limited. In CCK-8, clone formation and flow cytometry experiments, the proliferation of cells with low MED27/Sp1 expression was suppressed, while cell apoptosis was promoted. MED27 acted as an oncogene in BC. By affecting Sp1, MED27 could be a new therapeutic target for the treatment of BC.