Background: Stem cell therapy shows applications potential for malnutrition-induced ovarian failure in rat models. However, it is ineffective because of the lack of viability and differentiation of transplanted stem cells, resulting in low adaptation and survival rates. We aimed to determine whether stem cells cultured under low oxygen (O ₂) tension improves the adaptability and viability of stem cells, as well as ovarian failure. Methods: After four days of culturing mesenchymal stem cells (MSCs) in 21% oxygen (normoxia) as the T2 group and 1% oxygen (low O ₂ or hypoxia) as the T1 group, 200 million bone marrow-derived MSCs per rat were transplanted into female rats with ovarian failure (15 rats per treatment group). A total of 15 fertile and 15 infertile rats were categorized as the C+ and C- groups, respectively. Results: The slight increase in cells expressing HSP70 (C+, T2, T1, and C- groups were 0.5 ^a±0.53, 1.7 ^a±0.82, 6.2 ^b±1.5, and 9.6 ^c±1.3, respectively), decrease in cells expressing caspase-3 as an apoptotic inhibitor (C+, T2, T1, and C- groups were 0.2 ^a±0.42, 0.6 ^a±0.52, 4.8 ^b±1.03, and 7.3 ^c±1.42, respectively), and increase in cells expressing VEGF-1 (C+, T2, T1, and C- groups were 10.8 ^c±1.55, 8.7 ^b±0.48, 0.4 ^a±0.52, and 0.2 ^a±0.42, respectively) and GDF-9 (C+, T2, T1, and C- groups were 5.8 ^c±1.47, 4.6 ^b±0.97, 0.5 ^a±0.53, and 0.3 ^a±0.48, respectively) were used as markers for viability and differentiation in ovarian tissue, indicating that MSCs cultured under low O ₂ tension were more effective than those cultured under normoxic conditions as a treatment for female rats with ovarian failure. Furthermore, infertile female rats treated with MSCs cultivated under low O ₂ tension had an enhanced ovarian tissue shape, as indicated by the increasing Graafian follicle count (C+, T2, T1, and C- groups were 8.9 ^c±0.74, 4.5 ^b±0.71, 0.5 ^a±0.53, and 0.4 ^a±0.52, respectively). Conclusions: MSCs cultured under low O ₂ tension are an effective treatment for malnourished rats with ovarian failure.