Objective Currently, there is no cure for chronic pancreatitis (CP). Germline loss-of-function variants in SPINK1 (encoding trypsin inhibitor) are common in patients with CP and are associated with acute attacks and progression of the disease. This preclinical study was conducted to explore the potential of adeno-associated virus type 8 (AAV8)-mediated overexpression of human SPINK1 ( hSPINK1 ) for pancreatitis therapy in mice. Design A capsid-optimised AAV8-mediated hSPINK1 expression vector (AAV8- hSPINK1 ) to target the pancreas was constructed. Mice were treated with AAV8- hSPINK1 by intraperitoneal injection. Pancreatic transduction efficiency and safety of AAV8- hSPINK1 were dynamically evaluated in infected mice. The effectiveness of AAV8- hSPINK1 on pancreatitis prevention and treatment was studied in three mouse models (caerulein-induced pancreatitis, pancreatic duct ligation and Spink1 c.194+2T>C mouse models). Results The constructed AAV8- hSPINK1 vector specifically and safely targeted the pancreas, had low organ tropism for the heart, lungs, spleen, liver and kidneys and had a high transduction efficiency (the optimal expression dose was 2×10 11 vg/animal). The expression and efficacy of hSPINK1 peaked at 4 weeks after injection and remained at significant level for up to at least 8 weeks. In all three mouse models, a single dose of AAV8 -hSPINK1 before disease onset significantly alleviated the severity of pancreatitis, reduced the progression of fibrosis, decreased the levels of apoptosis and autophagy in the pancreas and accelerated the pancreatitis recovery process. Conclusion One-time injection of AAV8 -hSPINK1 safely targets the pancreas with high transduction efficiency and effectively ameliorates pancreatitis phenotypes in mice. This approach is promising for the prevention and treatment of CP.