To investigate the effects of trimethylamine-N-oxide (TMAO) enrichment on the survival and osmoregulation of Portunus trituberculatus under low salinity conditions, P. trituberculatus juveniles were fed diets containing different concentrations TMAO (0, 0.2%, 0.4% and 0.8%). Following a 15 day-TMAO enrichment period, the P. trituberculatus were subjected to low salinity (4) stress. The results showed that dietary TMAO supplementation increased the molting rate of P. trituberculatus under normal salinity (26). Specifically, the highest molting rate was observed in the 0.8% TMAO-supplemented group, reaching 53.9%±0.4% on day 15. All TMAO concentrations enhanced the survival rate of P. trituberculatus under low salinity stress. Notably, the 0.2% TMAO-enriched group exhibited the highest survival rate of 75% at 216 h of low salinity stress, which was significantly higher than the control group (P<0.05). Furthermore, the TMAO-enriched P. trituberculatus displayed higher serum osmotic pressure, gill Na⁺-K⁺ ATPase activity, mRNA gene expression of the α subunit of this enzyme, and TMAO content in various tissues compared to the control. This study suggests that TMAO enrichment may enhance the survival of P. trituberculatus under low salinity conditions by promoting molting and improving osmatic regulation through the use of inorganic ion and osmolytes. These findings provide a potential dietary supplement for the aquaculture of P. trituberculatus in low salinity environments.