For rainbow trout, hypoxia usually occurs during transportation and leads to deterioration of meat qualities. Hence, the study aimed to investigate the impacts of hypoxia during short-time transportation on the muscle quality of rainbow trout and its relief attempt by reoxygenation. In comparison with the sample without hypoxia (CG), the fillets from sever hypoxic trout (SHG) exhibited lower shearing force (SF), a*, saturated fatty acid content and delicious amino acids, and higher expressible moisture, Metmyoglobin (MetMb) and essential amino acids content. After subjected to reoxygenation for 24 h, the muscle qualities of trout recovered to the level of CG, except for expressible moisture and tissue structure. Metabolomic analyses indicated that hypoxia led to the up-regulation of succinate, xanthine, adenine and down-regulation of 15(S)-HETE ((15S,5Z,8Z,11Z,13E)-15-Hydroxyeicosatetraenoic acid). And the reoxygenation promoted glycine and serine biosynthesis, thiamine and cysteine metabolism and inhibited succinate accumulation, thus relieved the hypoxia. This work suggested that reoxygenation for 24 h was an effective action to relief the quality deterioration of rainbow trout fillets induced by hypoxia.