Background: Acute pancreatitis-activated Kupffer cells undergo accelerated apoptosis via NF-κB transcriptional regulation of Fas/FasL. We tested the hypothesis that Protein kinase C-zeta (PKC-ζ), which regulates cell death via NF-κB, plays a critical role in Kupffer cell apoptosis during acute pancreatitis. Methods: Acute pancreatitis was induced by cerulein injection in adult male Sprague-Dawley rats; 24 hrs later, liver homogenates were assayed for key cell signaling systems [PKC-ζ protein (immunoblots) and kinase activity, NF-κB nuclear translocation (ELISA), Fas/FasL (immunoblot)], and apoptosis [(Caspase-3 activation (immunoblot), DNA fragmentation (ELISA]. In an in-vitro model of acute pancreatitis, fresh rat Kupffer cells were infected with a PKC-ζ domain-negative adenovirus (AdPKCζ-DN) to inhibit PKC-ζ and then were treated with pancreatic elastase (1U/ml). Cellular extracts were assayed for PKC-ζ , NF -κB, Fas/FasL, Caspase-3, and DNA fragmentation. n≥ 3; data: mean±SD; ANOVA and t-test with Bonferroni’s correction were used. Results: Cerulein-induced pancreatitis upregulated PKC-ζ protein (3,305±18 vs. 1,555±38), PKC-ζ activity (2.4±0.1 vs. 1±0.1), NF-κB nuclear translocation (16±0.3 vs. 4±0.1), Fas (3,551±26 vs.1540±26), FasL (3,343±27 vs. 1,444±26), Caspase-3 activation (2,553±33 vs. 1,053±32) and DNA fragmentation (30±1 vs. 8±1%) in rat livers (all p<0.001 vs. control). In vitro, infection of fresh Kupffer cells with AdPKCζ-DN abolished the elastase-induced upregulation of PKCζ protein and activity, NF-κB nuclear translocation, Fas, FasL, Caspase-3 activation and DNA fragmentation (Table: all p < 0.001; * vs. control untreated cells; ¶ vs. infection control). Conclusion: Pancreatitis induces upregulation of PKC-ζ and Kupffer cell apoptosis. Inhibiting PKC-ζ significantly attenuated the elastase-induced upregulation of key pro-apoptotic signaling within Kupffer cells and reduces apoptosis. Regulation of Kupffer cell apoptosis by PKC-ζ and NF-κB warrants further investigation. The ability of Kupffer cells to autoregulate their stress response by apoptosis may have therapeutic implications.