AIM: To investigate the effects of glutathione-S-transferase (GST) gene polymorphism on the concentration of 6-thioguanine nucleotides (6-TGN), an active metabolite of azathioprine (AZA), in patients with inflammatory bowel disease (IBD), in order to provide reference for the optimization of AZA treatment in patients. METHODS: The clinical data of patients with IBD treated by AZA were collected prospectively, the genotypes of GST-A1, GST-M1, GST-P1 and GST-T1 were detected by targeted sequencing of multiplex PCR combined with high-throughput sequencing technology before administration, and the steady-state trough concentrations of 6-TGN in patients' red blood cells were determined by HPLC. Statistical analysis was carried out by SPSS 26.0 software. RESULTS: A total of 90 patients were included in this study. The alleles frequencies of GST-A1, GST-M1, GST-P1 and GST-T1 were consistent with Hardy-Weinberg equilibrium law. Logistic regression analysis showed that carrying GST-A1 mutant gene was an independent risk factor for the increase of trough concentration of 6-TGN (low concentration OR=17.50, P=0.030; high concentration OR=3.60, P=0.033), while the gene polymorphism of GST-M1, GST-P1, GST-T1 had no significant correlation with the concentration of 6-TGN (P>0.05). CONCLUSION: The gene polymorphism of GST-A1 may affect the concentration of 6-TGN, an active metabolite of AZA, and detection of GST-A1 genotype before AZA treatment will contribute to clinical individualized medication.