Tilapia tolerate hypoxia; thus, they are an excellent model for the study of hypoxic adaptation. In this study, we determined the effect of acute hypoxia stress on the antioxidant capacity, metabolism, and gill/liver ultrastructure of male genetically improved farmed tilapia (GIFT, Oreochromis niloticus). Fish were kept under control (dissolved oxygen (DO): 6.5 mg/L) or hypoxic (DO: 1.0 mg/L) conditions for 72 h. After 2 h of hypoxia stress, antioxidant enzyme activities in the heart and gills decreased, while the malondialdehyde (MDA) content increased. In contrast, in the liver, antioxidant enzyme activities increased, and the MDA content decreased. From 4 to 24 h of hypoxia stress, the antioxidant enzyme activity increased in the heart but not in the liver and gills. Cytochrome oxidase activity was increased in the heart after 4 to 8 h of hypoxia stress, while that in the gills decreased during the later stages of hypoxia stress. Hypoxia stress resulted in increased Na⁺-K⁺-ATP activity in the heart, as well as hepatic vacuolization and gill lamella elongation. Under hypoxic conditions, male GIFT exhibit dynamic and complementary regulation of antioxidant systems and metabolism in the liver, gills, and heart, with coordinated responses to mitigate hypoxia-induced damage.