A recent study reported that endothelial PAS domain protein 1 (EPAS1; hypoxia-inducible factor 2α) acts downstream of PGC-1α to regulate the slow-twitch muscle fiber program in mice, yet its role in human physiology and exercise adaptation remains unclear. The aim of this study was threefold: (1) to investigate EPAS1 gene expression in human skeletal muscle and its association with muscle fiber composition and the expression of endurance-related genes; (2) to determine how EPAS1 expression responds to aerobic training; and (3) to examine whether EPAS1 genetic variation is linked to aerobic capacity, hemoglobin, and athletic status. The study involved 1234 subjects, including 943 athletes and 291 untrained individuals. EPAS1 gene expression was significantly higher in endurance athletes compared with power athletes (p = 0.011) and was positively associated with the proportion of slow-twitch muscle fibers in the vastus lateralis of untrained subjects (p = 0.0008) and athletes (p = 0.0033). EPAS1 expression was higher in females (p = 0.0028) and negatively associated with smoking status (p = 0.0007). Moreover, EPAS1 expression showed positive association with endurance-related genes, including ANGPT2, CKM, CPT1B, EPOR, FNDC5, HIF1A, KDR, MYBPC3, NFATC4, NOS3, PPARA, PPARD, PPARGC1A, UCP2, and VEGFA. Analysis of 24 publicly available skeletal muscle transcriptomic datasets demonstrated that EPAS1 expression increased significantly (meta-analysis p = 9.2 × 10-5) following aerobic training. Finally, genetically predicted higher EPAS1 expression (i.e., carriage of the EPAS1 rs6756667 A allele) was positively associated with endurance athlete status in both sexes (p = 0.0004) and with VO₂max (p = 0.046) and hemoglobin (p = 0.041) in male athletes. These findings potentially identify EPAS1 as an important genetic factor associated with muscle fiber composition, endurance-related phenotypes, and adaptation to aerobic training.