OBJECTIVE To explore the protective effects and potential mechanisms of 1α, 25(OH)(2) D(3) (VitD(3)) on pancreatic β-cells. METHODS The apoptosis of NIT-1 cells was induced by interleukin-1β (IL-1β) and interferon-γ (IFN-γ) in vitro. Then the apoptotic rate of NIT-1 cells was determined by Hoechest33342 staining and Annexin V-FITC/PI flow cytometry. The insulin secretion level of NIT-1 cells was measured by ELISA. The NIT-1 cells were treated with VitD(3) at the final concentrations of 10(-8) mol/L or underwent transient transfection with vitamin D receptor (VDR)-SiRNA. RESULTS After the treatment of VitD(3), the apoptotic rate of NIT-1 cells decreased to 39.7%. There were significant differences in apoptotic rate between the VitD(3) treatment and IL-1β/IFN-γ groups (68.4%) (P < 0.01). Similarly impaired glucose-stimulated insulin secretion (GSIS) of NIT-1 cells recovered ((7.34 ± 0.21) ng/ml) after the treatment of VitD(3) as compared with the IL-1β/IFN-γ group ((4.88 ± 0.32) ng/ml, P < 0.01). Moreover, most of the protective effects of VitD(3) on pancreatic β-cells could be blocked by the transfection of VDR-SiRNA. CONCLUSION VitD(3) may protect pancreatic β-cells from cytokine-induced apoptosis and impaired insulin secretion through its conjugation with VDR.