Osteoporosis is a common disease that makes bones prone to fracture and can affect both men and women. Many traditional Chinese medicine formulations have the potential effect of preventing osteoporosis. Gu Ling Pian (GLP), a traditional Chinese medicine formulation, comprised of tonifying kidney herbal medicines, has been demonstrated to prevent osteoporosis by increasing bone mineral density, however the exact mechanism has not yet been elucidated. Osteoprotegerin (OPG), a receptor activator of NF-kappaB (RANK), and RANK ligand (RANKL) play critical roles in bone remodelling by regulating the function of osteoclasts. In this study, we investigated the effect of GLP on osteoblasts, namely MG-63 cells. The cell proliferation and differentiation, synthesis of OPG/RANKL and p38 expression were tested on MG-63 cells exposed to serum from rats fed with GLP or not. The results showed that GLP significantly promoted MG-63 cell proliferation and differentiation. Upregulation of OPG and down-regulation of RANKL at the protein and mRNA level were observed in GLP serum treated MG-63 cells using an enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Further, treatment with GLP serum increased the level of p38 phosphorylation but did not affect the total p38 expression. These effects can be blocked by the p38 specific inhibitor SB203580. The results indicate that GLP can effectively promote the proliferation and differentiation of osteoblasts and regulate their OPG/RANKL expression, while the effects may be mediated via the p38 MAPK pathway. The findings suggest that GLP induces bone formation and may be beneficial for patients with osteoporosis.