CD4+T cells mediate the pathogenesis of renal ischemia-reperfusion injury (IRI). Emerging research suggests that a Th17/regulatory T cell (Treg) imbalance plays a pivotal role in the development of renal IRI. The recently identified negative checkpoint T cell immunoglobulin domain and mucin domain family 3 (Tim-3) inhibits the immune response by binding to its ligand, galectin-9 (Gal-9). However, the role of the Gal-9/Tim-3 signaling pathway in the regulation of CD4+T cell subsets in renal IRI remains unclear. In this study, the effect of the Gal-9/Tim-3 signaling pathway on Th17/Treg subsets in renal IRI was investigated using a mouse model. Renal IRI induced the expression of Gal-9 in renal tubular epithelial cells and increased the percentages of Tim-3+Th17 cells and Tim-3+Foxp3+Treg cells in the IR kidneys. The administration of rAAV9-Gal-9 suppressed kidney inflammation, reduced the mortality of mice with renal IRI, increased Foxp3+Treg cells, and reduced Th17 cells. In contrast, the blockade of Tim-3 in vivo with an anti-Tim-3 mAb aggravated renal inflammation, decreased Foxp3+Treg cells, and promoted Th17 cells. Thus, Gal-9/Tim-3 signaling pathway activation may protect against renal IRI by inhibiting Th17 cell production and inducing Foxp3+Treg cell expansion. Our study suggests that the Gal-9/Tim-3 signaling pathway might become a target of immunotherapy in renal IRI.