Bone marrow-derived mesenchymal stem cells (BM-MSCs) have the capacity to differentiate into osteoblasts and adipocytes. Bone marrow adipogenesis exerts an inhibitory effect on osteogenesis, which leads to osteoporosis. S100A16, a novel member of the S100 family, is ubiquitously expressed, and markedly enhances adipogenesis. The aim of this study was to demonstrate, in the mouse BM-MSC model, whether S100A16 significantly stimulates adipogenic, rather than osteogenic differentiation. The overexpression of S100A16 led to a significant increase in Oil Red O staining (a marker of adipocyte differentiation) but a decrease in Alizarin Red S staining (a marker of osteoblast differentiation). In contrast, reducing the expression of S100A16 resulted in minimal Oil Red O staining but increased Alizarin Red S staining. During differentiation into osteoblasts, RUNX2 expression increased fourfold in the S100A16(KO+/-) BM-MSCs, but only increased by approximately 1.5-fold in the S100A16(TG+/+) BM-MSCs. And BMP2 occurred in the same changes. Upon induction of BM-MSC differentiation into adipocytes, peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein-α expression were significantly higher in the cells overexpressing S100A16 protein but lower in the cells with reduced expression of S100A16 protein, compared with the control cells, which were BM-MSCs derived from C57/BL6. S100A16 increased PPARγ promoter luciferase activity and decreased RUNX2 promoter luciferase activity. ERK1/2 phosphorylation was stimulated during osteogenesis, whereas p-JNK phosphorylation was increased by stimulation of adipogenesis. Our results suggest that S100A16 inhibits osteogenesis but stimulates adipogenesis by increasing the transcription of PPARγ and decreasing the transcription of RUNX2. The ERK1/2 pathway is involved in the regulation of osteogenesis whereas the JNK pathway is involved in adipogenesis.