Ugonin K is a flavonoid isolated from the roots of Helminthostachys zeylanica, a folk medicine used to strengthen bone mass and cure bone fracture. It is of interest to determine whether ugonin K has beneficial effect on osteoblast maturation. In this study, MC3T3-E1 osteoblasts were treated with ugonin K. Cell differentiation and mineralization were identified by alkaline phosphatase (ALP) activity and Alizarin red S staining, respectively. RT-PCR and Western blot were used to analyze osteoblast-associated gene expression and signaling pathways. Our results showed that ugonin K significantly induced the increase of ALP activity, expressions of bone sialoprotein (BSP) and osteocalcin (OCN), and mineralization. The mRNA expressions of the transcription factors Runx2 and osterix were also up-regulated by ugonin K. Ugonin K increased the phosphorylated level of p38 and ERK, respectively. In the presence of SB203580, ugonin K induced expressions of Runx2 and osterix, ALP activity, BSP level and bone nodule formation were all completely inhibited, but ugonin K induced OCN expression was not affected. On the other hand, ugonin K-induced ALP activity and mineralization were mildly attenuated by PD98059, but the over-expressed Runx2, osterix, BSP and OCN also were significantly repressed by PD98059. These suggested that both p38 and ERK participate in regulating ugonin K evoked osteogenesis but p38 seemed to play a more important role. Take together, the potential anabolic effect of ugonin K on bone might act through activations of p38- and ERK-mediated Runx2 and osterix expressions to induce the synthesis of osteoids and formation of bone nodule.