Hypoxia, a common environmental condition, can affect cell survival and physiological function by triggering oxidative stress. Akt/FoxO pathway has been proven to play a non-negligible role in the regulation of autophagy. However, the role of Akt/FoxO pathway in hypoxia-induced autophagy is unclear in fish. Therefore, in this study, grass carp hepatocyte cells (L8824) were treated by CoCl2 to simulate hypoxia, and the results showed that CoCl2 can increase the expression of Hif-1α protein at different concentrations or different treatment time. Further study found that hypoxia increased intracellular reactive oxygen species (ROS) level, and the expression of autophagy-related genes (LC3-II, pink1, beclin-1 and p62) and foxO1a/1b. The mitochondrial membrane potential (Δψm) was also depolarized, and autophagosomes were intriguingly detected by transmission electron microscope (TEM) after the treatment of hypoxia. Moreover, hypoxia inhibited Akt phosphorylation, while PI3K/Akt pathway inhibitor, LY294002 significantly up-regulated the expression of foxO1a/1b and autophagy-related genes. Additionally, silencing foxo1b also resulted in down-regulation of autophagy-related genes. It was demonstrated that hypoxia induced autophagy via Akt/FoxO1 pathway. These results will provide a new light on further understanding the role of Akt/FoxO pathway in the response to hypoxia in fish.