Oxygen is necessary for fish survival, and low oxygen levels affect the growth, reproduction, and survival of fish. miR-462 and miR-731 are located in the same gene cluster (miR-462-731), which is unique to teleost fish; the expression of this gene cluster is significantly upregulated in hypoxic environments. Mitochondria are the centers of energy metabolism and use oxygen for aerobic respiration to power the body. The function of miR-462-731 is speculated to be related to the mitochondria. To investigate whether the miR-462-731 cluster could regulate mitochondrial function in grass carp (Ctenopharyngodon idella), we analyzed the effects of miR-462 and miR-731 overexpression on the biological function of mitochondria in grass carp liver cells. The mRNA expression levels of key enzyme genes in the tricarboxylic acid cycle were detected by qRT-PCR after the overexpression of miR-462 and miR-731, and the expression of mdh, ogdh and cs significantly decreased. Simultaneously, the ATP content was significantly reduced. The JC-1 probe was used to detect the mitochondrial membrane potential of cells after transfection. The content of reactive oxygen species (ROS) in the cells increased significantly, whereas the total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity decreased significantly; however, the content of malondialdehyde (MDA) increased. Electron microscopy revealed that the overexpression of miR-462 and miR-731 led to mitochondrial structural damage. In conclusion, this study found that overexpression of miR-462-731 could reduce cellular energy metabolism and the antioxidant capacity of grass carp liver, destroy mitochondrial structure, and affect mitochondrial function. This study further improved the functional study of miR-462-731 and provided a theoretical basis for the hypoxic regulation mechanism and hypoxic tolerance of grass carp and other bony fishes.