We previously reported that Prunella vulgaris aqueous extract (PVAE) promotes hepatic glycogen synthesis and decreases postprandial hyperglycemia in ICR mice. Inflammatory cytokines play a critical role in the pathogenesis of diabetes. This study was designed to examine whether PVAE has a protective effect on IL-1β-induced apoptosis in INS-1 cells. INS-1 pancreatic β cells were plated at 2×10 6/ml and treated with PVAE (100 μg/ml) 30 min before the cells were challenged with IL-1β (10 ng/ml). Untreated INS-1 cells served as control. INS-1 cell cytotoxicity was examined by MTT and lactate dehydrogenase (LDH) activity assays. Caspase-3 activity and activation of the apoptotic signaling pathway were analyzed by western blotting. NF-κB binding activity was examined by EMSA. The levels of inflammatory cytokines in the supernatant were measured by ELISA. IL-1β treatment significantly induced INS-1 cell death by 49.2%, increased LDH activity by 1.5-fold and caspase-3 activity by 7.6-fold, respectively, compared with control cells. However, PVAE administration significantly prevented IL-1β-increased INS-1 cell death and LDH activity and attenuated IL-1β-increased caspase-3 activity. Western blot data showed that PVAE also significantly attenuated IL-1β-increased Fas, FasL and phospho-JNK levels in the INS-1 cells. In addition, PVAE treatment significantly attenuated IL-1β-increased NF-κB binding activity and prevented IL-1β-increased TNF-α and IL-6 expression in INS-1 cells. Our data suggest that PVAE has a protective effect on IL-1β-induced INS-1 cell apoptosis. PVAE also attenuates IL-1β-increased NF-κB binding activity and inflammatory cytokine expression in INS-1 cells. PVAE may have a benefit for type I diabetic patients.