Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65* Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. We used HEK 293 cells stably expressing GPR65 and deficient for either Gα s , Gα q/11 or Gα 12/13 , to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. We confirmed that GPR65 signals via the Gα s pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα 12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1β secretion in response to an inflammatory stimulus. We demonstrate that GPR65 exerts its effects through Gα s - and Gα 12/13 -mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα 12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies. [Display omitted] • In response to low pH, GPR65 signals via Gαs, leading to cAMP accumulation. • In response to low pH, GPR65 signals via Gα12/13, leading to RhoA activation. • The IBD-associated GPR65*231Leu variant shows reduced affinity to Gα12/13. • GPR65 is involved in the process of phagocytosis and actin remodeling in macrophages. • GPR65 regulates inflammasome activation and the pro-inflammatory cytokine response. [ABSTRACT FROM AUTHOR]