Seleno-β-lactoglobulin (Se-β-Lg) is a selenium conjugating protein synthesized by binding β-lactoglobulin (β-Lg) with inorganic selenium. The present study was designed to investigate the antitumor mechanism of Se-β-Lg on human gastric cancer MGC-803 cells. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), scanning electron microscope (SEM), Annexin V-FITC/PI double staining and cell cycle detection suggested that Se-β-Lg exhibited a significant inhibitory effect on the proliferation of MGC-803 cells with typical morphological characteristics of apoptosis by inducing cell cycle arrested at G2/M phase. Additionally, Se-β-Lg could induce the disruption of mitochondrial membrane potential (MMP), improve the levels of intracellular reactive oxygen species and Bax, and down-regulate the Bcl-2 expression, further resulting in the release of cytochrome c from mitochondria into cytoplasm, the activation of caspase-9/-3, and the cleavage of poly-ADP-ribose polymerase (PARP). Taken together, these data clearly indicated that Se-β-Lg had significantly cytotoxic effects on MGC-803 cells by inducing the caspase-dependent cell apoptosis and triggering the Bax- and Bcl-2-mediated mitochondria apoptosis pathway.