Ischemia reperfusion injury remains a significant challenge in various clinical settings, necessitating effective therapeutic interventions. Hypothermia has emerged as a promising strategy for mitigating cellular damage associated with oxygen deprivation. Our study investigates the molecular mechanisms underlying hypothermia-mediated protection using an in vitro model system. We demonstrate that hypothermia exerts a dual effect on cellular metabolism, simultaneously suppressing metabolic activity while enhancing stress tolerance. Specifically, mild hypothermia at 32°C emerges as the optimal temperature for balancing these opposing effects.Mechanistically, hypothermia attenuates hypoxia-induced metabolic disturbances, including alterations in lactate:pyruvate ratios and gene expressions associated with reductive stress. Notably, our research reveals a previously unrecognized role of hypothermia in modulating gene expression, with over 3000 genes responding to cooling, surpassing the response to hypoxia. Intriguingly, hypothermia-induced activation of hypoxia response element (HRE) transcriptional activity is dependent on HIF1B, independent of HIF1A or HIF2A.At 32°C, hypothermia activates Nrf2, promoting thioredoxin and glutathione-related gene expression and elevating glutathione levels. Furthermore, pre-conditioning with hypothermia enhances cellular resilience against oxidative stress.Current evidence highlighting potentially therapeutic molecular mechanisms can be translated to in vivo models, for further evaluation of therapeutic efficacy. Understanding the interplay between hypothermia, metabolic regulation, and stress response pathways holds promise for developing novel therapeutic strategies to alleviate ischemia reperfusion injury.