Osteoblastic differentiation and bone‐forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3‐E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3‐E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time‐ and concentration‐dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3‐E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3‐E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3‐E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways.