Cissus quadrangularis Linn. has been implicated as therapeutic agent for enhancing bone healing. Though its osteogenic activity has been suggested, the underlying mechanism still remains unclear. In the present study, the effects of ethanol extract of C. quadrangularis (CQ-E) on osteoblast differentiation and function were analyzed using murine osteoblastic cells. The results indicated that mRNA expressions of osteoblast-related genes were not affected by the CQ-E treatment. However, alkaline phosphatase (ALP) activity and the extent of mineralized nodules were significantly increased in treated cells compared with controls. The addition of an extracellular regulated kinase 1/2 inhibitor, a Jun N-terminal kinase 1/2/3 inhibitor and a p38 mitogen-activated protein kinase (MAPK) inhibitor resulted in significantly decreased ALP activity, preferentially by p38 MAPK inhibitor. These results suggested that CQ-E may regulate osteoblastic activity by enhancing ALP activity and mineralization process, and the increased ALP activity effect of CQ-E is likely mediated by MAPK-dependent pathway.