S100 calcium-binding protein B (S100B) is expressed and released by adipocytes, and is positively correlated with body mass index, however, the direct effects of S100B on adipocytes remain unclear. Bone marrow‑derived mesenchymal stem cells have the capacity to differentiate into osteoblasts and adipocytes, which is important for bone metabolism. The current study aimed to determine the effect of S100B on adipogenesis and osteogenesis. The mouse embryo cell line C3H/10T1/2 was used to build cell models with varying levels of S100B protein expression. Western blot analysis was performed to assess the expression of various marker proteins. Oil red O staining and alizarin red S staining were used to detect adipogenesis and osteogenesis, respectively. S100B overexpression was associated with a significant increase in oil red O staining and a significant reduction in alizarin red S staining. Runt‑related transcription factor‑2 and bone morphogenetic protein 2 expression levels were significantly increased in the S100B underexpression group, however not in the S100B overexpression group. By contrast, the expression levels of the adipogenesis markers peroxisome proliferator‑activated receptor γ and CCAAT‑enhancer‑binding protein α was significantly increased in the S100B overexpression group, however not in the S100B underexpression group. Osteogenesis stimulation increased extracellular signal‑regulated kinase (ERK) phosphorylation, and adipogenesis stimulation increased c‑Jun N‑terminal kinase (JNK) phosphorylation. The results suggest that S100B inhibits osteogenesis, however stimulates adipogenesis. The ERK pathway is involved in the regulation of osteogenesis, whereas the JNK pathway is involved in the regulation of adipogenesis.