The large yellow croaker (Larimichthys crocea) is one of the most economically important marine fish species in China. Reducing water salinity can effectively prevent white spot disease, but it can also cause low-salinity stress and damage to fish. Low-salinity acclimation can improve the body’s tolerance to low salinity; therefore, it is necessary to explore the specific physiological mechanisms of low-salinity acclimation in fish under low-salinity stress. This study aimed to investigate the effects of low-salinity acclimation on energy metabolism and mitophagy in the large yellow croaker under low-salinity stress. Juvenile large yellow croakers with a body weight of (53.46±1.47) g were transferred from natural seawater (salinity 25) to water with a salinity of 20 and were cultured for 7 d and then exposed to low-salinity (salinity 12) water for 24 h. The results showed that low-salinity stress increased reactive oxygen species (ROS), adenosine triphosphate (ATP) content, tricarboxylic acid (TCA) cycle enzyme activities, and mitophagy gene expression levels, indicating that L. crocea improved aerobic metabolism and mitophagy under low-salinity stress but still suffered from oxidative damage. Compared with the low-salinity group, the low-salinity acclimation with low-salinity group showed increased ATP content, TCA enzyme activities, and mitophagy gene expression levels but reduced ROS content, indicating that low-salinity acclimation reduced oxidative damage by improving energy metabolism and mitophagy in large yellow croaker under low-salinity stress. During low-salinity stress, adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activity was positively correlated with TCA and fatty acid β oxidation enzyme activities and negatively correlated with fatty acid synthesis enzyme activity, indicating that AMPK promoted energy-yielding metabolism and inhibited energy-consumptive metabolism in L. crocea, thereby improving energy generation efficiency. Forkhead box class O3 (FoxO3) mRNA levels were positively correlated with the expression levels of mitophagy genes, indicating that FoxO3 participates in the regulation of mitophagy gene expression during low-salinity adaptation. In summary, low-salinity acclimation improved aerobic metabolism and mitophagy, inhibited lipid synthesis, and enhanced the tolerance of L. crocea to low-salinity stress.