Heart failure (HF) remains a major health concern without a definitive treatment. Although survival rates have overall improved, the currently available therapeutics are palliative and cannot restore cardiac tissue. Autologous stem cell therapy using cardiac progenitor cells (CPCs) has shown promising results in clinical trials by reducing scar size and restoring cardiac function in post-MI hearts. The overall improvement has not been significant enough to translate into further clinical trials, therefore, the approach requires refinement. Major challenges are poor retention and survival. Conventionally, the isolation and expansion procedure of CPCs is done under ambient air containing 21% O2 prior to injection. Stem cells, however, have been shown to reside in hypoxic niches in vivo and retain their function in hypoxic conditions in vitro as a consequence of avoiding damaging effects of reactive oxygen molecules. The goal of this study is to elucidate how 1% O2 isolation and expansion impacts CPC self-renewal, survival and metabolic function in comparison to regular cell culture. I hypothesized that hypoxic culture would protect CPCs from senescence by maintaining mitochondrial function. Human HF patients CPCs were isolated and expanded side by side in permanent hypoxia (1% O2) or in normoxia (21% O2). Hypoxic CPCs showed improved self-renewal, reduced senescence, oxidative damage, and enhanced survival in response to H2O2. Higher mitochondrial function in hypoxic CPCs was observed, possibly due to increased NAD+ /NADH ratio and improved autophagic mitochondrial quality control. Taken together, these data demonstrate that permanent hypoxic culture is beneficial to the CPCs, and that early senescence observed in normoxic CPCs is driven by mitochondrial dysfunction.