<p>Mammals dwelling at different altitudes exhibit distinct molecular mechanisms to adapt to low-oxygen environments owing to habitat-specific oxygen levels. Notably, these adaptations include energy metabolism patterns, which fundamentally sustain vital physiological functions. Skeletal muscle, a pivotal contributor to systemic energy metabolism, facilitates vertebrate body movement through the contraction and relaxation of muscle fibers and is highly dependent on mitochondrial substrate oxidation for energy production. This study focused on three rodent species inhabiting different altitudes: the Qinghai vole (<i>Neodon fuscus</i>), Brandt’s vole (<i>Lasiopodomys brandtii</i>), and Kunming mouse (<i>Mus musculus</i>). Using transcriptomics and quasi-targeted metabolomics, we systematically analyzed the differences in skeletal muscle metabolic regulation among the three rodent species before and after exposure to hypoxia, thereby revealing the underlying molecular mechanisms. In summary, <i>N. fuscus</i>, native to high-altitude environments, tended to sustain energy supplies through regulating fatty acid oxidation under low-oxygen conditions. Conversely, <i>L. brandtii</i> and <i>M. musculus</i>, acclimatized to middle- and low-altitude habitats, relied on aerobic oxidation and anaerobic glycolysis of glucose, respectively, for energy maintenance under hypoxic conditions. In addition to their differential metabolic preferences under hypoxic conditions, these three rodent species showed species-specific responses related to oxygen utilization, antioxidant defense mechanisms, and anti-inflammatory processes. This study provides insights into the metabolic response patterns of mammalian skeletal muscle under hypoxic conditions, thereby establishing a basis for future investigations on transcriptional–metabolic associations.</p> Graphical abstract <p></p>