Background Pectoralis phenotypic variation plays a fundamental role in locomotion and thermogenesis in highland birds. However, its regulatory and metabolic mechanisms remain enigmatic to date. Here, we integrated phenomic, transcriptomic and metabolomic approaches to determine muscle variation and its underpinning mechanisms across altitudinal songbirds. Results Phenomics revealed that all highland birds had considerable increases in muscle oxidative capacity, capillarity, and mitochondrial abundance. Correspondingly, transcriptomic analyses found that differentially expressed genes in modules associated with phenotypes enriched in blood vessel, muscle structure development, and mitochondrial organization. Despite similar traits and functional enrichments across highland birds, different mechanisms drove their occurrence in part for their own various evolutionary histories. Importantly, a metabolic feature shared by highland birds is the improvement in insulin sensitivity and glucose utilization through activating insulin signaling pathway, which is vital to increase muscle oxidative capacity and maintain metabolic homeostasis. Nevertheless, fatty acid biosynthesis and oxidation are enhanced in species with a long evolutionary history, also differing from ketone body metabolism in recently introduced colonizer. Conclusions Our study represents a vital contribution to reveal the regulatory and metabolic basis of pectoralis variation across altitudinal songbirds.