Abstract: Background Impairment of pancreatic islet β cell function is one of the main pathological mechanisms of type 2 diabetes mellitus (T2DM). Serotonin (5-hydroxytryptamine, 5-HT) secreted by pancreatic islet β cells themselves can increase the number of pancreatic islet β cells and promote insulin secretion under glucose stimulation. Jiao-Tai-Wan (JTW) is a traditional Chinese medicine formula with hypoglycemic effect. However, it is currently unclear whether JTW improves impaired pancreatic islet β cell function by regulating the pancreatic 5-HT system. Objective To explore the hypoglycemic effect of JTW in db/db mice and its specific molecular mechanism. Methods JTW solution was prepared, and its main active ingredients were identified by high-performance liquid chromatography. All db/db mice were randomly divided into 5 groups with 8 mice in each, namely: diabetic model group (Mod group, intragastric administration of equal volume of distilled water), metformin group (Met group, intragastric administration at 0.25 g ·kg^-1·d^-1), low-dose JTW group (JTWL group, intragastric administration at 2.1 g ·kg^-1·d^-1), medium dose JTW group (JTWM group, intragastric administration at 4.2 g ·kg^-1·d^-1), and high-dose JTW group (JTWH group, intragastric administration at 8.4 g ·kg^-1·d^-1). The experiment lasted for 6 weeks. The body weight and fasting plasma glucose (FPG) of mice were measured weekly. After 6 weeks, blood was collected from the canthus, and the mice were sacrificed by cervical dislocation. Pancreatic tissue morphology was evaluated by hematoxylin-eosin staining. The serum insulin level was detected by enzyme-linked immunosorbent assay (ELISA). The messenger RNA (mRNA) expression levels of Ins1 and Ins2 in pancreatic tissue were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The expression of indicators related to the pancreatic 5-HT system, including 5-HT, 5-hydroxytryptamine receptor 2B (5-HTR_2B), and transient receptor potential ankyrin 1 (TRPA1) was evaluated by ELISA, qRT-PCR, immunofluorescence, or immunohistochemistry. Results JTW reduced the FPG levels of db/db mice, but had no significant effect on their body weight. Meanwhile, JTW improved the morphological structure of pancreatic islet cells, increased the serum insulin concentration, and up-regulated the mRNA expression levels of Ins1 and Ins2 in pancreatic tissue. In addition, JTW also up-regulated the expression of 5-HT, 5-HTR_2B, and TRPA1 in the pancreatic tissue of db/db mice. Conclusion JTW can improve pancreatic islet β cell function by regulating the pancreatic 5-HT system, which provides a new research idea for clarifying the specific molecular mechanism of JTW in the treatment of T2DM.