Acute pancreatitis (AP) is currently among the most prevalent digestive diseases. The pathogenesis of AP remains elusive, and there is no specific treatment. Therefore, identifying novel therapeutic targets is imperative for effective management and prevention of AP. In this study, we conducted a comprehensive transcriptomic analysis of peripheral blood from patients with AP and the pancreatic tissue from a mouse model of AP. Our analyses revealed that mouse model of AP exhibited a higher enrichment of mitogen-activated protein kinase signaling, endocytosis, apoptosis and tight junction pathways than the control. Subsequent weighted gene co-expression network analysis identified 15 gene modules, containing between 50 and 1000 genes each, which demonstrated significant correlations within samples from patients with AP. Further screening identified four genes (ACSL4, GALNT3, WSB1, and IL1R1) that were significantly upregulated in severe acute pancreatitis (SAP) in both human and mouse samples. In mouse models of SAP, ACSL4 was significantly upregulated in the pancreas, whereas GALNT3, WSB1, and IL1R1 were not. Lastly, we found that a commercially available ACSL4 inhibitor, PRGL493, markedly reduced IL-6 and TNFα expression, alleviated pancreatic edema and necrosis, and diminished the infiltration of inflammatory cells. In conclusion, this study comprehensively depicts the key genes and signaling pathways implicated in AP and suggests the potential of ACSL4 as a novel therapeutic target for SAP. These findings provide valuable insights for further exploration of therapeutic strategies for SAP.