Prostate cancer is among the most prevalent malignancies in men. High intake of dairy products is associated with an increased risk of prostate cancer. No study has examined the gene profile changes and molecular mechanism by which casein, milk protein, affects prostate cancer cells. In this study, we used gene expression profiling to identify gene changes in PC3 prostate cancer cells exposed to α-casein. α-casein altered the expression of a large number of genes-related prostate cancer, transcription factor, apoptotic, metabolic, and cell cycle pathways, in addition to the expected cell proliferation signaling pathways. To clarify the molecular events involved in the effect of α-casein on proliferation and progression of PC3 cells, we examined cell proliferation assay, quantitative real-time PCR, Western blotting, and immunohistochemical and immunofluorescence staining. α-casein promoted PC3 cell proliferation and survival under serum-free conditions by increasing the expression of E2F1 and its target gene PCNA. α-casein also protected PC3 cells from serum-starved autophagic cell death by activating the PI3K/Akt pathway through activation of mTORC1, up-regulation of p70S6K, and down-regulation of LC3 autophagosome formation. Our data provide new insights into the molecular mechanisms underlying the tumorigenic effect of α-casein in prostate cancer cells.