The protein hydrolysates of fishes have been reported to be a potential source of many health benefits components for pharmaceutical or nutritional applications. The aim of this study is to examine the possible antiproliferative function of roe protein hydrolysates of Alburnus tarichi using enzymatic hydrolysis against breast cancer cells and explore its detailed mechanisms. In addition, we evaluated the effects of protein hydrolysate on the proliferation and apoptosis of two human breast cancer cell lines (MCF-7 and MDA-MB-231). The cultured cells were treated with protein hydrolysate at concentrations of 0-5 mu g/ml for 24 h and 48 h. Inhibition of cell proliferation, percentage of apoptotic cells, cell cycle distribution, morphological changes, DNA fragmentation, intracellular reactive oxygen species (ROS) production, and apoptotic protein levels were also examined. Decreases in proliferation of MCF-7 and MDA-MB-231 cells were observed after treatment with the protein hydrolysate in a dose-dependent manner. Distinct morphological changes, a typical pattern of fragmented DNA, and increased intracellular ROS production and apoptotic protein levels were observed in both cell lines after hydrolysate treatment (p < 0.05). The results suggested that the protein hydrolysate inhibits the proliferation of human breast cancer cell lines by introducing apoptosis through a caspase-dependent pathway in a dose-dependent manner.