Drug residues from aquaculture have been associated with negative effects on human and environmental health. The in vivo metabolism of a drug is carried out by a number of enzymes and can influence the formation of drug residues or the drug effect. However, few studies have evaluated the effects of drugs used in aquaculture on drug metabolism enzymes in marine crustaceans. Glutathione S-transferases play an important role in drug metabolism. We isolated and characterized glutathione S-transferase gene in Portunus trituberculatus, a species that is widely cultured in the offshore waters of China. The cDNA encoding GST was first cloned using RT-PCR and Smart-Race. Analysis of the nucleotide sequence revealed that the full-length cDNA clone was 1 010 bp and encoded a protein of 216 amino acids, which had a predicted molecular weight of 24.59 kD with an estimated PI of 5.294. The protein was expected to possess the GST-N structure domain and GST-C structural domain, suggesting that it belonged to the GST subgroup. Comparison of amino acid sequences showed a similarity of more than 78% between GST in P. trituberculatus and Eriocheir sinensis. The phylogenetic analysis revealed that the GST in P. trituberculatus was in the same branch as that of E. sinensis. The level of GST gene expression in different tissues was analyzed by quantitative real-time PCR. GST was detected in all tested tissues of P. trituberculatus, including the hepatopancreas, gills, muscle, hemolymph, and eyestalk. Expression was highest in the hepatopancreas (P<0.05) and lowest in the heart. GST expression was up-regulated in the hepatopancreas after intramuscular injection of sulfadiazine (P<0.05). This suggests that GST expression was induced by the exogenous drug and increased the capacity for detoxification of the organism. GST expression decreased with time and was significantly(P<0.05) lower than controls between 12 and 18 h post injection. This suggests that the function of hepatopancreas cells was disrupted. However, expression of the GST gene increased significantly between 24 h and 48 h, suggesting that detoxification of hepatopancreas cells was restored. Our results suggest that GST may play a role in the drug-metabolic response of P. trituberculatus.