Ellagic acid has been shown to inhibit tumor cell growth. However, the underlying molecular mechanismsremain elusive. In this study, our aim was to investigate whether ellagic acid inhibits the proliferation of MCF-7 human breast cancer cells via regulation of the TGF-β/Smad3 signaling pathway. MCF-7 breast cancer cellswere transfected with pEGFP-C3 or pEGFP-C3/Smad3 plasmids, and treated with ellagic acid alone or incombination with SIS3, a specific inhibitor of Smad3 phosphorylation. Cell proliferation was assessed by MTTassay and the cell cycle was detected by flow cytometry. Moreover, gene expression was detected by RT-PCR,real-time PCR and Western blot analysis. The MTT assay showed that SIS3 attenuated the inhibitory activityof ellagic acid on the proliferation of MCF-7 cells. Flow cytometry revealed that ellagic acid induced G0/G1 cellcycle arrest which was mitigated by SIS3. Moreover, SIS3 reversed the effects of ellagic acid on the expression ofdownstream targets of the TGF-β/Smad3 pathway. In conclusion, ellagic acid leads to decreased phosphorylationof RB proteins mainly through modulation of the TGF-β/Smad3 pathway, and thereby inhibits the proliferationof MCF-7 breast cancer cells.