In this study, the apoptosis induction and antitumor activity of a novel complex, seleno-β-lactoglobulin (Se-β-Lg), on H22 cells were explored. In in vitro experiments, the MTT assay showed that Se-β-Lg was cytotoxic to H22 cells in a concentration- and time-dependent manner and displayed few proliferation inhibition effects on normal liver L02 cells. Annexin V-FITC/PI and PI staining assays showed that Se-β-Lg induced apoptosis changes of H22 cells from early to late apoptosis and led to S phase cell cycle arrest. Western blot and Z-VAD-FMK inhibitor assays showed that Se-β-Lg triggered the Fas/FasL-mediated caspase 8-dependent extrinsic death receptor pathway in H22 cells. In in vivo experiments, Se-β-Lg effectively repressed the growth of transplanted H22 solid tumors in a dose-dependent manner and exhibited few toxic effects on the host animals. H&E and PI staining of tumor tissues showed that Se-β-Lg caused the occurrence of typical apoptosis morphology features and dose-dependently increased the proportion of apoptosis peaks (Sub-G1 peak) in H22 solid tumors. These results suggest that Se-β-Lg has the capacity to induce H22 tumor cell apoptosis in vitro and in vivo and support that Se-β-Lg can be applied as a functional complex in food.