Background and Aims ATP citrate lyase (Acly) is widely expressed in various tissues and has been shown to play a crucial role in many inflammatory diseases. However, the role of Acly in acute pancreatitis (AP)-induced pancreatic injury remains unclear. This study was conducted to investigate the effect of Acly on pancreatic injury in a mouse AP model and to preliminarily explore its underlying mechanism.Methods Twelve male C57BL6/J Acly^cKO mice (conditional pancreatic Acly knockout) and twelve Acly^wt mice (with intact pancreatic Acly expression) were randomly divided into AP groups (administered caerulein at 100 μg/kg intraperitoneally, 6 times/d, with 1-hour intervals, for 4 consecutive days) and control groups (administered saline in the same manner), with six Acly^cKO mice and six Acly^wt mice in each group. Pancreatic tissue samples were collected 24 hours after modeling to observe pathological changes in the pancreas and to measure the expression of the macrophage marker CD68, the neutrophil marker myeloperoxidase (MPO), as well as nuclear factor NF-κB p65, Toll-like receptor 4 (TLR4), and pro-inflammatory factors TNF-α and IL-1β.Results Compared with the control group, the AP group exhibited significant pathological changes in the pancreas. The pathological damage in Acly^cKO mice was more severe than in that in Acly^wt mice, with significantly higher inflammation and necrosis of pancreatic tissue scores in Acly^cKO mice (all P<0.05). In the AP group, the pancreatic expressions of CD68 and MPO were significantly higher in Acly^cKO mice than those in Acly^wt mice (both P<0.05). Additionally, Acly^cKO mice showed increased nuclear positivity for NF-κB p65 and elevated expression of TLR4, TNF-α, and IL-1β compared with Acly^wt mice (all P<0.05).Conclusion Acly exerts a protective effect against AP-induced pancreatic injury, possibly by inhibiting the activation of the NF-κB signaling pathway, thereby mitigating the inflammatory response.