Abstract: Under the combined pressures of global warming and water eutrophication, offshore salmonid aquaculture systems face increasingly threats from hypoxia during summer due to the expansion of oceanic anoxic zones. Landlocked rainbow trout (Oncorhynchus mykiss) and anadromous steelhead (Oncorhynchus mykiss) as the main culture species in deep-sea cage may exhibit different degrees of hypoxic tolerance caused by different life histories. To investigate the effects of chronic hypoxia on growth, serum biochemical, liver antioxidant enzyme activity, and related gene expression of rainbow trout and steelhead, we established four treatment groups as follows: rainbow trout normoxia group (R-CT), rainbow trout hypoxia group (R-HY), steelhead normoxia group (S-CT), and steelhead hypoxia group (S-HY). At the conclusion of 30d trial, our results demonstrated that chronic hypoxia significantly reduced the percent weight gain, total feed, specific growth rate, and thermal-unit growth coefficient in both rainbow trout and steelhead. However, there were no significant differences in growth observed between the two fish species. Following hypoxic stress, serum glucose (GLU) levels decreased while lactate dehydrogenase (LDH) activity increased in rainbow trout; whereas serum GLU levels increased and LDH activity decreased in steelhead. This suggests that liver glycolysis in rainbow trout was activated to meet energy demands under hypoxia, while it was inhibited in steelhead. Furthermore, levels of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) in rainbow trout serum, as well as serum ALP in steelhead, significantly decreased hypoxia, indicating substantial metabolic responses in both species. The activities of catalase (CAT) in rainbow trout liver, as well as superoxide dismutase (SOD) and CAT in steelhead liver, significantly increased under low-oxygen conditions, indicating superior ability to neutralize reactive oxygen species (ROS) in steelhead compared to rainbow trout. The integrated biomarker (IBR) index showed significant divergence among the different groups, with the R-HY group (6.61) > S-HY group (5.94) > S-CT (0.43) > R-CT groups (0.37), indicating inferior survival of rainbow trout compared to steelhead under hypoxia condition. The up-regulation of vascular endothelial growth factor (VEGF) in rainbow trout liver, as well as VEGF and glucose transporter 1 (GLUT1) in steelhead liver, in response to hypoxia suggests slightly different hypoxic responses in the HIF-1 signaling pathway of both fish species. In summary, this study demonstrated that rainbow trout are more sensitive to hypoxia (4.08±0.45) mg/L than steelhead, and significant physiological response were observed between the two salmonids life forms.