Osteoblasts have the capacity to differentiate into several different cell types, including adipocyte, chondrocyte, and muscle lineages. Therefore, osteoblast can be potentially applied in the treatment of bone diseases. The factors controlling osteoblast differentiation is complex. Recently, it has been reported that some natural products regulate the differentiation in osteoblasts and promote bone formation. Based on these findings, this study demonstrated that Lycium barbarum polysaccharides (LBP) could promote proliferation of osteoblast MC3T3‐E1 cells through 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Besides, expression of key proteins correlated with cellular proliferation such as proliferating cell nuclear antigen (PCNA) and Ki67 was enhanced in the presence of LBP. We also detected the increased expression of bone‐specific matrix proteins such as morphogenetic protein 2 (BMP2), bone Gla protein (BGP), osteopontin (OPN), and α‐1 type‐I collagen (COL1A1) when treated with LBP. This process was mediated by some signals, such as smad1, smad8, Runt‐related transcription factor 2 (RUNX2), and Osterix. Furthermore, RUNX2 silencing inhibited osteoblast differentiation by decreasing expression of bone‐specific matrix proteins. Collectively, we proposed a previously unidentified function of LBP in osteoblast differentiation, suggesting its potential clinical role in bone disease treatment.