Background: Organisms living at high altitudes face low oxygen and temperature conditions; thus, the genetic mechanisms underlying the adaptations in these organisms merit investigation. The glyptosternoid fish, Creteuchiloglanis macropterus mainly inhabits regions with gradual increases in altitudes along the Nujiang River and might serve as an appropriate evolutionary model for detecting adaptation processes in environments with altitude changes. Results: We constructed eleven RNA-sequencing (RNA-seq) libraries of C. macropterus collected from five locations at different altitudes to identify the genetic signatures of high-altitude adaptation. The comparative genomic analysis indicated that C. macropterus has an accelerated evolutionary rate compared with that of fishes in the lowland, and fishes at higher altitudes might evolve faster. Functional enrichment analysis of the fast-evolving and positively selected genes, differentially expressed genes and highly expressed genes, showed that these genes were involved in many functions related to energy metabolism and hypoxia. Conclusions: Our study provides evidence of high-altitude adaptation in C. macropterus, and the detected adaptive genes might be a resource for future investigations of adaptations to high-altitude environments in other fishes.