Metformin is one of the most commonly used drugs for type 2 diabetes mellitus. In addition to its anti-diabetic property, evidence suggests more potential applications for metformin, such as antiaging, cellular protection, and anti-inflammation. Studies have reported that metformin activates pathways with anti-inflammatory effects, enhances the integrity of gut epithelial tight junctions, and promotes a healthy gut microbiome. These actions contribute to the protective effect of metformin against gastrointestinal (GI) tract injury. However, whether metformin plays a protective role in psychological-stress-associated GI tract injury remains elusive. We aim to elucidate the potential protective effect of metformin on the GI system and develop an effective intervention strategy to counteract GI injury induced by acute psychological stress. By monitoring the levels of GI-nonabsorbable Evans blue dye in the bloodstream, we assessed the progression of GI injury in live mice. Our findings demonstrate that the administration of metformin effectively mitigated GI leakage caused by psychological stress. The GI protective effect of metformin is more potent when used on wild-type mice than on activating-transcription-factor 3 (ATF3)-deficient (ATF3−/−) mice. As such, metformin-mediated rescue was conducted in an ATF3-dependent manner. In addition, metformin-mediated protection is associated with the induction of stress-induced GI mRNA expressions of the stress-induced genes ATF3 and AMP-activated protein kinase. Furthermore, metformin treatment-mediated protection of CD326+GI epithelial cells against stress-induced apoptotic cell death was observed in wild-type but not inATF3−/−mice. These results suggest that metformin plays a protective role in stress-induced GI injury and that ATF3 is an essential regulator for metformin-mediated rescue of stress-induced GI tract injury.