Abstract: Eriocheir sinensis is an economically important crab species endemic to China. It has a migratory life history. At the megalopa stage, the larvae migrate from seawater to freshwater. In artificial breeding, a gradual desalination (salinity reduction) process is commonly applied to facilitate the successful transition of juveniles to freshwater environments. To shorten the freshwater acclimation period, the physiological responses of E. sinensis to an abrupt decrease in salinity were investigated. A total of 1 000 healthy megalopa were randomly collected from a seawater aquaculture pond (salinity 26) and temporarily acclimated under identical conditions for 3 d, then they were divided into a seawater group (SW) and a freshwater group (FW). Differences in survival rate, gill structure, energy metabolism, and tryptophan metabolism of megalopa were compared after 24 h between the SW group (cultured in seawater with salinity 26) and the FW group (transferred directly from seawater with salinity 26 to freshwater with salinity 0). The results showed that the survival rate of megalopa was not affected after acute low-salinity stress. Extensive sloughing of the gill cuticle was observed, accompanied by a reduction in hemocytes in the gill filaments and lamellae. Whole-body Na⁺/K⁺-ATPase activity increased extremely significantly (P < 0.01), and immunohistochemical staining revealed enhanced localization of Na⁺/K⁺-ATPase in the gill lamellae. Glycogen content decreased extremely significantly (P < 0.01), whereas glucose content increased extremely significantly(P < 0.01), and crustacean hyperglycemic hormone (CHH) content increased significantly (P < 0.05). Whole-body tryptophan hydroxylase (TPH) mRNA expression increased extremely significantly (P < 0.01), and TPH1 showed intensified staining in the ommatidia of the compound eye of megalopa. A total of 17 tryptophan (Trp) metabolism-related metabolites differed significantly before and after stress. Among them, indole-3-acetic acid (IAA), nicotinic acid (Na), and picolinic acid (PA) contents decreased significantly (P < 0.05), while indole-3-carboxaldehyde (IAld) content decreased extremely significantly (P < 0.01). In contrast, tryptamine (TRM) content increased significantly (P < 0.05), and kynurenine (KYN) content increased extremely significantly (P < 0.01). In conclusion, the megalopa of the E. sinensis exhibits strong tolerance to acute low-salinity stress, as their survival rate was not affected after direct transfer from seawater to freshwater for 24 h. Megalopa respond to acute low-salinity stress by regulating energy metabolism (upregulating CHH levels and enhancing Na⁺/K⁺-ATPase activity) and tryptophan metabolism (upregulating TPH expression and altering Trp-derived metabolites in the kynurenine and indole pathways). This study may provide a theoretical basis for shortening the acclimation time of megalopa of the E. sinensis to low-salinity conditions.