Milk is a potential nutraceutical with wide range of bioactive compounds that are antioxidative， antimicrobial， antithrombotic， immunomodulatory， opioid and antihypertensive. Various intervention studies with milk reflect its stupendous role in elevating bone mineral density. Milk and milk products have shown a preventive effect in bone loss during pre- and postmenopausal women. Since， milk is proved to have a vital role in bone health promotion， there is a need to identify bioactive compounds within it. Recently we have reported four novel peptides from milk casein for their osteoblast proliferation activity. Their role in differentiation and the signaling cascade evoked by them have not been studied. Thus， the present study has been designed to investigate the differentiation potential and signaling cascade of one of the novel peptides， that is， NAVPITPTL by analyzing osteoblast differentiation markers such as alkaline phosphatase activity， osteocalcin and mineral deposition. All the experimentations suggested a significant role of this peptide in osteoblast differentiation. The inhibitor studies， immunocytochemistry and immunoblotting have proven that the peptide-induced differentiation through pAkt signaling cascade as pAkt was observed in nucleus. Moreover， the peptide was found to be bioaccessible up to 1%.