Nine major cell populations among 46,716 cells were identified in mouse intestinal ischemia‒reperfusion (II/R) injury by single-cell RNA sequencing. For enterocyte cells, 11 subclusters were found, in which enterocyte cluster 1 (EC1), enterocyte cluster 3 (EC3) and enterocyte cluster 8 (EC8) were newly discovered cells in ischemia 45 min/reperfusion 720 min group. EC1 and EC3 played roles in digestion and absorption, and EC8 played a role in cell junctions. For TA cells, after ischemia 45 min/reperfusion 90 min, many TA cells at the stage of proliferation were identified. For Paneth cells, Paneth cluster 3 was observed in the resting state of normal jejunum. After ischemia 45 min/reperfusion 90 min, three new subsets were found, in which Paneth cluster 1 had good antigen presentation activity. The main functions of goblet cells were to synthesize and secrete mucus, and a novel subcluster (goblet cluster 5) with high proliferative ability was discovered in ischemia 45 min/reperfusion 90 min group. As a major part of immune system, the changes in T cells with important roles were clarified. Notably, enterocyte cells secreted Guca2b to interact with Gucy2c receptor on the membranes of stem cells, TA cells, Paneth cells and goblet cells to elicit intercellular communication. One marker known as glutathione S-transferase mu 3 (GSTM3) affected intestinal mucosal barrier function by adjusting MAPK signaling during II/R injury. The data on the heterogeneity of intestinal cells, cellular communication and the mechanism of GSTM3 provides a cellular basis to treat II/R injury.