The balance between bone formation and bone resorption is maintained by osteoblasts and osteoclasts, and an imbalance in this bone metabolism leads to osteoporosis. Here, we found that osteoblast differentiation in MC3T3-E1 cells is promoted by the inactivation of O-linked β-N-acetylglucosaminidase (O-GlcNAcase) and suppressed by the inactivation of O-GlcNAc transferase, as indicated by extracellular matrix calcification. The expression of osteogenic genes such as alp, ocn, and bsp during osteoblast differentiation was positively regulated in a O-GlcNAc glycosylation-dependent manner. Because it was confirmed that Ets1 and Runx2 are the two key transcription factors responsible for the expression of these osteogenic genes, their transcriptional activity might therefore be regulated by O-GlcNAc glycosylation. However, osteoclast differentiation of RAW264 cells, as indicated by the expression and activity of tartrate-resistant acid phosphatase, was unaffected by the inactivation of either O-GlcNAcase or O-GlcNAc transferase. Our findings suggest that an approach to manipulate O-GlcNAc glycosylation could be useful for developing the therapeutics for osteoporosis.