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Dissolved oxygen (DO) plays a crucial role in survival, growth, and normal physiological functions of aquatic organisms. Nevertheless, the mechanisms involved in hypoxic stress and adaptation have not been fully elucidated in Largemouth bass (Micropterus salmoides). To reveal the effect of acute hypoxia on Largemouth bass, we simulated acute hypoxia (DO: 1.2 ± 0.2 mg/L) in the laboratory and analyzed physiological parameters (RBCs, Hb, SOD, CAT, NA+/K+-ATPase, GPx, and MDA) and gene expression (HIF-1alpha and GLUT-1) in Largemouth bass exposed to various durations of acute hypoxia (0, 1, 2, 4, 8, 12, and 24 h). Our results indicated that acute hypoxic exposure significantly increased RBCs but decreased Hb. In addition, antioxidant enzyme activity was enhanced significantly in the liver and muscles at the initial stage of acute hypoxic exposure, but decreased significantly in gills during the entire process of hypoxic exposure. Furthermore, the expression levels of HIF-1alpha and GLUT-1 mRNA were significantly up-regulated in Largemouth bass under acute hypoxic exposure. In conclusion, our study provides a valuable basis for further elucidation of hypoxic adaptation and facilitates husbandry for an economically valuable species.RBCs was increased in Largemouth Bass at initial hypoxic exposure.Antioxidant enzymes were increased in liver and muscle under hypoxic exposure.Antioxidant enzymes were decreased in gill during hypoxic exposure.The HIF-1alpha and GLUT-1 mRNA expression was up-regulated after hypoxic exposure.