Monoclonal antibody therapy for immune checkpoint blockade has achieved promising results for several types of malignant tumors. For the future treatment of gastrointestinal stromal tumors (GISTs) by immune checkpoint blockade, expression of immune checkpoint-related molecules that suppress antitumor immunity in GISTs was examined. Infiltration of immune cell types into 19 GIST tissues was analyzed by immunohistochemistry, and expression of T cell immunoglobulin and mucin protein 3 (Tim-3) and programmed cell death-1 (PD-1) in the infiltrated immune cells was examined by immunofluorescence microscopy. The expression status of galectin-9 in the GIST tumor cells was also determined by immunohistochemistry. All the GIST tissues showed CD8+ T cell infiltration and 8 showed CD56+ natural killer (NK) cell infiltration, and the numbers of infiltrated CD8+ T and NK cells were strongly correlated. However, these CD8+ T and NK cells were CD69-negative inactivated cells. Tim-3 was expressed in the infiltrated NK cells in 6/8 (75%) of the GIST tissues. Expression of galectin-9, a ligand of Tim-3, was observed in 13/19 (68.4%) GIST tissues and all of the GIST tissues with Tim-3+ NK cell infiltration showed positive galectin-9 expression. No PD-1 expression in the infiltrated NK cells and neither Tim-3 nor PD-1 expression was observed in the infiltrated CD8+ T cells. Interaction between Tim-3 in infiltrated NK cells and galectin-9 in tumor cells may be involved in an immune checkpoint mechanism for suppression of antitumor immunity in GISTs. Blockade of the Tim-3/galectin-9 pathway may become a new strategy for GIST treatment.