AIM: To examine the impact of the transcription factor 7 analogue 2 (TCF7L2) gene polymorphism on the hypoglycaemic effect of exenatide in patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 100 newly diagnosed Han Chinese patients with T2DM who had not received any drug treatment were selected from the Affiliated Hospital of Xuzhou Medical University and treated with exenatide monotherapy for 6 months. The TCF7L2 rs290487 was genotyped by SnaPshot method, and blood glucose levels, lipids profiles and pancreatic function evaluation indicators were measured at baseline, 3 months and 6 months after exenatide treatment. Multiple linear regression analysis was employed to assess the correlation between each indicator and the reduction in glycated hemoglobin (HbA1c) levels after 6 months of exenatide treatment. The expression of TCF7L2 protein in the plasma of T2DM patients was detected by enzyme-linked immunosorbent assay (ELISA) kit. Furthermore, western blotting was conducted to ascertain TCF7L2 expression in pancreatic tissues obtained from db/db mice and INS-1 cells cultured under high glucose conditions. Lentivirus transfection was used to overexpress or knock down TCF7L2 in insulinoma cell line (INS-1) cells, followed by measurement of KSIS activity and insulin content after a 24-hour intervention with exenatide. RESULTS: The distribution pattern of TCF7L2 rs290487 was found to be in accordance with Hardy-Weinberg equilibrium (P>0.05). Following 6 months of exenatide treatment, there was a notable reduction in blood glucose levels and an improvement in lipid profiles when compared to baseline values. Additionally, there was a significant increase in the homeostasis model assessment of beta-cell function (HOMA-B) values. Patients with the TT genotype exhibited significantly lower postprandial plasma glucose (PPG) levels and HbA1c values compared to those with the CC or CT genotypes (P<0.05). After adjusting for age, gender, body mass index (BMI), and waist to hip ratio (WHR) in the multiple linear regression model, a significant association was observed between the rs290487 TT genotype, baseline HbA1c levels, and family history of diabetes with the reduction in HbA1c after six months of exenatide treatment (P<0.05). Furthermore, individuals with the rs290487 TT genotype demonstrated a notable elevation in TCF7L2 expression in plasma among T2DM patients in comparison to those with the CC genotype (P<0.05). In particular, pancreatic tissue from db/db mice exhibited markedly elevated TCF7L2 expression compared to [db/][m] mice. However, this up-regulation was reversed by exenatide treatment. Similarly, INS-1 cells cultured under high glucose conditions demonstrated an increase in TCF7L2 expression, which was ameliorated upon exenatide administration. The knockdown of TCF7L2 using shRNA enhanced the KSIS function of pancreatic β cells and augmented the insulinotropic effect of exenatide. Conversely, the upregulation of TCF7L2 impaired the KSIS function of pancreatic β cells and attenuated the insulinotropic effect of exenatide. CONCLUSION: The TCF7L2 rs290487 gene polymorphism is closely associated with the hypoglycaemic efficacy of exenatide therapy. The risk allele C may diminish the effectiveness of exenatide by impacting the levels of PPG and HbA1c in T2DM patients. The mutation at TCF7L2 rs290487 site (C→T) influenced the expression of TCF7L2 protein. By exerting its regulatory effect, exenatide may be capable of regulating the impact of TCF7L2 on the function of pancreatic β cells.