Waterborne ammonia is a threat to animal health and its accumulation is typical of aquatic ecosystems. Autophagy serves as a safeguard of intracellular homeostasis, yet its role in maintaining the health of hemocytes, the master regulators of crustacean immunity, remains unclear. Herein, the swimming crab (Portunus trituberculatus) is employed as a case study to illustrate the impact of ammonia on hemocyte health via autophagy. This study showed the occurrence of abnormal cellular structure and significant accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS) (P <
0.05), demonstrating that severe ammonia stress can damage hemocytes. This was accompanied by significant increase of autophagy hemocytes fraction and apoptosis (P <
0.05). Meanwhile, there was a significant increase in the expression of Beclin1 and microtubule-associated protein 1 light chain 3 (LC3-II) (P <
0.05). This suggests an ammonia-induced autophagy initiation. However, ammonia stress significantly decreased lysosomal fluorescence intensity (P <
0.05) and expression of the marker gene lysosomal-associated membrane protein 1 (LAMP1) (P <
0.05). These imply an ammonia-induced repression of lysosome-dependent autophagy degradation, which may underlie the pronounced increase in apoptosis (P <
0.05). After the administration of the autophagy activator rapamycin (Rap), rather than the inhibitor 3-Methyladenine (3-MA), the levels of apoptosis, ROS and the fraction of autophagic cells were significantly decreased (P <
0.05), demonstrating a mitigation of the ammonia-induced cell damage through lysosome-dependent autophagy degradation. This study sheds light on how crustaceans respond to ammonia exposure by demonstrating the significance of lysosome-dependent autophagy in maintaining hemocyte health.