Background: Among the numerous well-documented medicinal herbs, Eurycoma longifolia (EL) has gained remarkable recognition due to its promising efficacy of stimulating bone formation in androgen-deficient osteoporosis. Though numerous animal studies have explored the bone-forming capacity of EL, the exact mechanism was yet to be explored. Objective(s): The present study was aimed to investigate the mechanism of bone-forming capacity of EL using MC3T3-E1 as an in vitro osteoblastic model. Materials and methods: The cell differentiation capacity of EL was investigated by evaluating cell growth, alkaline phosphatase (ALP) activity, collagen deposition and mineralization. Taken together, time-mannered expression of bone-related mediators which include bone morphogenic protein-2 (BMP-2), ALP, runt-related transcription factor-2 (Runx-2), osteocalcin (OCN), type I collagen, osteopontin (OPN), transforming growth factor-beta 1 (TOE-beta 1) and androgen receptor (AR) were measured to comprehend bone-forming mechanism of EL. Results: Results demonstrated a superior cell differentiation efficacy of EL (particularly at a dose of 25 mu g/mL) that was evidenced by dramatically increased cell growth, higher ALP activity, collagen deposition and mineralization compared to the testosterone. Results analysis of the bone-related protein biomarkers indicated that the expression of these mediators was well-regulated in EL-treated cell cultures compared to the control groups. These findings revealed potential molecular mechanism of EL for the prevention and treatment of male osteoporosis. Conclusion: The resulting data suggested that EL exhibited superior efficacy in stimulating bone formation via up-regulating the expression of various mitogenic proteins and thus can be considered as a potential natural alternative therapy for the treatment of osteoporosis. (C) 2017 Transdisciplinary University, Bangalore and World Ayurveda Foundation. Publishing Services by Elsevier B.V.