The Notch signaling pathway has been implicated in tumor growth in a number of different human tissues, including the colon, liver, and breasts. Regulation of this pathway in breast tissue has been investigated through several different methods with limited success. This study explored the efficacy of short hairpin RNA (shRNA) in modulating Notch signaling through its downstream transcription factor RBPJ in the human breast cancer cell line MCF-7. In this cell line, shRNA delivery through a lentivirus vector effectively downregulated RBPJ expression and protein levels. Importantly, overall cell proliferation was significantly reduced, and there was an increase in p21 expression and decreases in the expression of CDK2, Hes1, bcl-2, and SKP2. Together, these data suggest that shRNA knockdown of RBPJ in the Notch signaling pathway could be a novel therapeutic approach to treating human breast cancer.