Effects of whey protein on osteoblasts were evaluated. The whey protein was added to the culture medium at concentrations of 0.02 and 0.1 mg/mL. In vitro, whey protein stimulated the proliferation and differentiation of osteoblasts cultured in different concentrations of whey protein. The levels of osteocalcin and insulin-like growth factor-I in the culture medium also increased. Real-time reverse transcription-PCR results showed that the mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL) increased in the cells in a dose-dependent manner, and when the results were expressed as OPG/RANKL ratio, a significant increase could be seen in the 0.1 mg/mL whey protein group. These results showed that the active component in the whey protein plays an important role in bone formation and a potential therapeutic role in osteoporosis by activating osteoblasts.