Ischemia/reperfusion (I/R) injury is responsible for liver injury during hepatic resection and liver transplantation. The plasma membrane-bound G protein-coupled bile acid receptor (TGR5) could regulate immune response in multiple liver diseases. Nevertheless, the underlying role of TGR5 in hepatic I/R injury remains largely unknown. This study aimed to investigate the potential mechanism of TGR5 in hepatic I/R injury. Wild-type (WT) and TGR5 knockout (TGR5KO) mice were used to perform hepatic I/R, and macrophages were isolated from mice for in vitro experiments. The results demonstrated that knockout of TGR5 in mice significantly exacerbated liver injury and inflammatory response. TGR5KO mice infused with WT macrophages showed relieved liver injury. Further study revealed that TGR5 knockout inhibited sirtuin 3 (SIRT3) and forkhead box O3 (FOXO3) expression. In vitro experiments indicated that SIRT3 inhibited acetylation, ubiquitination and degradation of FOXO3. FOXO3 inhibited HIF-1α transcription by binding to its promoter. TGR5 knockout inhibited SIRT3 expression, thus promoted the acetylation, ubiquitination, and degradation of FOXO3, which resulted in increased HIF-1α transcription and macrophages proinflammatory response. Collectively, TGR5 plays a critical protective role in hepatic I/R injury. FOXO3 deacetylation mediated by SIRT3 can attenuate hepatic I/R injury. TGR5 deficiency aggravates hepatic I/R injury via inhibiting SIRT3/FOXO3/HIF-1α pathway.