Inflammatory bowel disease (IBD) remains a complex and multifaceted condition, with its management dependent on a thorough understanding of its underlying mechanisms. While the ANXA1-FPR axis is implicated in the pathogenesis of IBD, its relationship with the NLRP3 inflammasome in this context has not been established. Thus, this study aimed to elucidate the intricate relationship between ANXA1, FPRs, and the NLRP3 inflammasome in the pathogenesis of IBD. For this purpose, mRNA and protein expression of ANXA1, FPRs, and NLRP3 inflammasome were analyzed using transcriptomic data (GSE179285) and immunohistochemistry on ileum and colon samples from CD patients and healthy controls. In vitro, ANXA1-derived peptide Ac(2-26)'s effect was tested on TNF-alpha-activated Caco-2 cells. Transcriptome analysis of GSE179285 revealed significantly increased levels of ANXA1, FPR1, FPR2, and NLRP3 transcripts in inflamed CD segments compared to controls. Immunohistochemistry confirmed these findings, showing strong immunoreactivity for ANXA1, FPR2, and NLRP3 in CD samples, particularly in the intestinal epithelium and inflammatory infiltrate. In vitro, ANXA1 and NLRP3 were co-expressed in Caco-2 cells stimulated with TNF-alpha. The peptide Ac(2-26) at 2 ng/mL significantly increased caspase-1 and IL-1beta levels, enhancing NLRP3 inflammasome activation. Ac(2-26) also reduced ROS production and preserved epithelial integrity by enhancing occludin and cadherin expression. Overall, this study highlights the crucial role of ANXA1-FPR axis in regulating inflammation in CD and its potential therapeutic implications. Ac(2-26) modulates NLRP3 inflammasome activation and oxidative stress, preserves epithelial barrier integrity, and shows promise as a therapeutic target for IBD treatment.