Uterine fibroids, or leiomyoma, are the most common benign tumors in women of reproductive age. Herein, the effect of silencing the mediator complex subunit 12 (Med12) gene in human uterine fibroid cells was evaluated. The role of Med12 in the modulation of Wnt/β-catenin and cell-proliferation-associated signaling was evaluated in human uterine fibroid cells. Med12 was silenced in the immortalized human uterine fibroid cell line (HuLM) using a lentivirus-based Med12 gene-specific RNA interference (RNAi) strategy. HuLM cells were infected with lentiviruses carrying Med12-specific short shRNA sequences or a non-functional shRNA scrambled control with green fluorescence protein (GFP). Stable cells that expressed low levels of Med12 protein were characterized. Wnt/β-catenin signaling, sex steroid receptor signaling, cell-cycle associated, and fibrosis associated proteins were measured. Med12 knockdown cells showed significantly (p<0.05) reduced levels of Wnt4 and β-catenin proteins as well as cell proliferation, as compared to scrambled control cells. Med12 knockdown cells also showed reduced levels of cell-cycle associated cyclin D1, Cdk1, and Cdk2 proteins as well as reduced activation of p-ERK, p-AKT, and TGFβ signaling pathways as compared to scrambled control cells. Moreover, TGFβ regulated fibrosis-related proteins such as fibronectin, collagen type 1, and PAI-1 were significantly (p<0.05) reduced in Med12 knockdown cells as compared to scrambled control cells. Together, these results suggest that Med12 play a key role in the regulation of HuLM cell proliferation through the modulation of Wnt/β-catenin, cell-cycle associated, and fibrosis-associated protein expression.