Objective: To investigate the effect of forkhead box C2 (FOXC2) gene silencing by siRNA on mRNA and protein expression of cancer stem cell (CSC) marker CD44 in breast cancer. Methods: Breast cancer MCF-7 cells were firstly cultured by routine methods, and then were cultured in mammosphere-forming conditions to enrich and screen out breast cancer CSCs. Next, the obtained CSCs were transfected with the lentiviral vector containing FOXC2-siRNA or negative control-siRNA, and CSCs transfected with empty vector were served as blank control. The FOXC2 mRNA and protein expressions in breast cancer CSCs were determined by real time RT-PCR and Western blot method, respectively, to assess the silencing efficacy, and then CD44 mRNA and protein expressions in each group of breast cancer CSCs were measured by real time RT-PCR and Western blot, respectively. All results were analyzed by comparing to the expression levels of blank control. Results: The breast cancer CSCs were successfully cultured from MCF-7 cells by mammosphere formation. Compared with breast cancer CSCs transfected with negative control siRNA, both FOXC2 mRNA and protein expressions were decreased significantly (P=0.00509, P=0.00001), and both CD44 mRNA and protein levels were down-regulated significantly in FOXC2-siRNA transfected breast cancer CSCs (P=0.00848, P=0.00218). Conclusion: FOXC2 gene silencing can inhibit the CD44 mRNA and protein expression in breast cancer CSCs. So, FOXC2 signaling pathway may possibly participate in the proliferation and differentiation process of breast cancer CSCs through regulating CD44 expression.