Hydrogen sulfide (H₂S) has been reported to be involved in the signaling of the inflammatory response; however there are differing views as to whether it is pro- or anti-inflammatory. In this study, we sought to determine if endogenously synthesized H₂S via cystathionine--lyase (CSE) plays a pro- or anti-inflammatory role in caerulein-induced pancreatitis. To investigate this, we used mice genetically deficient in CSE to elucidate the function of CSE in caerulein induced acute pancreatitis. We compared the inflammatory response and tissue damage of wild type (WT) and CSE KO mice following ten hourly administrations of 50 μg/kg caerulein or saline control. From this we found that the CSE KO mice showed significantly less local pancreatic damage as well as acute pancreatitis-associated lung injury in comparison to the WT mice. There were also lower levels of pancreatic eicosanoid and cytokines as well as reduced acinar cell NF-B activation in the CSE KO mice when compared with WT mice. Additionally, in WT mice, there was a greater level of pancreatic CSE expression and sulfide synthesizing activity in caerulein-induced when compared to the saline control. When comparing the two saline treated control groups, the CSE KO mice showed significantly less pancreatic H₂S synthesizing activity relative to the WT mice. These results indicate that endogenous H₂S generated by CSE plays a key pro-inflammatory role via NF-B activation in caerulein-induced pancreatitis and its genetic deletion affords significant protection against acute pancreatitis and associated lung injury.