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β-sitosterol derived from Clinacanthus nutans Lindau was tested for its in vitro osteogenic activity using MC3T3-E1 pre-osteoblasts. Our results indicated that β-sitosterol was non-toxic to the cells cultured at a concentration <20 μg/mL. Treatment of the cells with β-sitosterol significantly enhanced the alkaline phosphatase activity up to 210 and 204.6% at 5 and 10 μg/mL, respectively (P < .05). Similarly, the mineralization activity of the β-sitosterol treated cells was elevated up to 134, 168, 118% at a concentration of 2.5, 5, and 10 μg/mL, respectively (P < .05). In addition, this compound up-regulated several marker genes for osteoblast differentiation, including runx2, osx and col I to 2, 2.5 and 5.6 folds at 10 μg/mL, respectively (P < .05). The expression of p38 and ERK proteins involved in the MAPK signal pathway related to mineralization and differentiation was also enhanced. Thus, the osteoblastogenic activity of β-sitosterol was fully illustrated for the first time. [ABSTRACT FROM AUTHOR]