Human dental pulp stem cells (hDPSCs) are a source for cell therapy. Before implantation, an in vitro expansion step is necessary, with the inconvenience that hDPSCs undergo senescence following a certain number of passages, loosing their stemness properties. Long-term in vitro culture of hDPSCs at 21% (ambient oxygen tension) compared with 3-6% oxygen tension (physiological oxygen tension) caused an oxidative stress-related premature senescence, as evidenced by increased β-galactosidase activity and increased lysil oxidase expression, which is mediated by p16 pathway. Furthermore, hDPSCs cultured at 21% oxygen tension underwent a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16 and BMI-1 might play a role in the oxidative stress-associated premature senescence. We show that it is important for clinical applications to culture cells at physiological pO to retain their stemness characteristics and to delay senescence.