Crohn's disease (CD), a complex gastrointestinal disorder, can be attributed to a combination of genetic factors, immune system dysfunction, and environmental triggers. Aquaporin 9 (AQP9) has been implicated in immunoregulation and inflammation in various conditions, yet its function in CD remains unclear. Herein, we investigated the contribution of AQP9 to CD pathogenesis and its impact on inflammation and pyroptosis. Bioinformatic analysis showed a significant increase in AQP9 expression (above 2.5-fold change) in CD patients compared to controls. In vitro experiments using human colonic epithelial cells (HT-29) demonstrated that AQP9 inhibition attenuated lipopolysaccharide (LPS)-induced cell damage, inflammatory cytokine secretion, and pyroptosis. Mechanistically, AQP9 silencing suppressed NLRP3 inflammasome activation, suggesting a role in regulating pyroptosis. AQP9 silencing inhibited p38 MAPK phosphorylation, indicating a direct involvement in modulating this inflammatory pathway. Furthermore, our findings indicate that AQP9 exacerbates inflammation and pyroptosis via activating the p38 MAPK signaling pathway, known to contribute to CD pathogenesis. In vivo studies using a murine model of CD-like colitis revealed that AQP9 inhibition led to about 45% reduction in colitis severity scores and about 30% decrease in the production of inflammatory cytokine by inactivating NLRP3 inflammasome and the p38 MAPK signaling. To sum up, our study highlights the involvement of AQP9 in CD pathogenesis through modulation of inflammation and pyroptosis via the NLRP3 inflammasome and p38 MAPK signaling pathway. Targeting AQP9 may offer a promising therapeutic approach for CD by suppressing inflammatory responses and preventing tissue damage.