PurposeThe high morbidity and mortality associated with type 2 diabetes mellitus (T2DM) pose a significant global health challenge, necessitating the development of more efficient anti-diabetic drugs with fewer side effects. This study investigated the intervention of vitamin D3 combined with glibenclamide in rats with T2DM to elucidate its effects on pancreatic β-cells through the NF-κB pathway. MethodsTwenty-four healthy male Sprague-Dawley (SD) rats were randomly assigned to four groups: the control group (CG), the model group (MG), the glibenclamide group (GG), and the glibenclamide + vitamin D3 group (GDG). After inducing the T2DM model using high-fat and high-sugar diet and intraperitoneal injection of streptozotocin, the rats in the GG group were administered glibenclamide orally (0.6 mg/kg/day), while those in the GDG group received both glibenclamide (0.6 mg/kg/day) and vitamin D3 (500 IU/kg/day) in corn oil for a duration of 8 weeks. Biochemical indices were measured, and histopathological changes in pancreatic tissue and islet β cells were observed using hematoxylin and eosin staining. The expression of pancreatic nuclear factor κB (NF-κB), islet β-cells, and inflammatory cytokines were assessed using the TUNEL method and PCR. ResultsAccording to the data from this current study, the GDG group showed significant positive differences in plasma biochemical indices, as well as in the expression of β cells, NF-κB p65, TNF-α, IL-1β, INF-γ, and Fas, compared to the GG and CG groups (P < 0.05). ConclusionThe results suggest that vitamin D has beneficial effects on T2DM by improving the functions of islet β cells through inhibition of the NF-κB signaling pathway. Therefore, it is suggested that vitamin D supplementation, when used alongside antidiabetic drugs, may more effectively prevent and treat T2DM.