Tuftelin, an acidic protein, thought to play a role in the initial stages of ectodermal enamel mineralization, has since been detected in mesenchymal-derived tissues. During bone/cartilage development and regeneration, mesenchymal stem cells (MSCs) undergo an avascular period in a hypoxic environment, involving induction of hypoxia-inducible factor 1-alpha (HIF-1-alpha), a key component in this process. In the present study we investigated, in a mouse mesenchymal C3H10T1/2 stem cell model, the hypothesis that oxygen stress modulates tuftelin 1 expression in relation to HIF-1-alpha (Hif1a), in a mouse mesenchymal C3H10T1/2 stem cell model. The results of the present study showed a biphasic induction of tuftelin, similar to the pattern of HIF-1-alpha expression, in MSCs subjected to a hypoxic insult of 1% O2 through a period of 2–24 h. Immunocytochemistry analysis of the cells exposed to hypoxic insult for 2–24 h revealed the same biphasic pattern of tuftelin protein expression. Tuftelin localization appears to be mainly in the cytoplasm, and concentrated at the perinuclear region of the cells by 24 h of hypoxic insult. Based on our previous studies using the neuronal PC12 cell model, in which tuftelin induction was mediated by Hif1a, we propose that tuftelin is a member of oxygen-sensitive genes and implicated in the adaptive mechanisms regulating MSC function.