INTRODUCTION: During early pregnancy, fetal placental tissue implants into maternal decidual tissue, forming a unique interface where maternal immune cells do not reject the invading fetal cells. Given the roles of Galectin-9 and Tim-3 in tumor immune regulation, studying their distribution and function at this interface may provide insights into recurrent pregnancy loss. METHODS: This study uses single-cell transcriptomics, spatial transcriptomics, and multiplex immunohistochemistry to examine the expression and localization of Galectin-9 and TIM-3. Hormone-induced decidualization of immortalized human endometrial stromal cells was conducted to investigate Galectin-9 expression. RESULTS: The major immune cells in the maternal decidua, such as T cells, NK cells, and macrophages, co-express Galectin-9 and TIM-3. Unlike TIM-3, Galectin-9 is also highly expressed in endothelial cells and decidualized stromal cells. Among placenta-derived cells, Hofbauer cells (HBs) and Placenta-associated maternal monocytes/macrophages (PAMMs) exhibit high expression of both Galectin-9 and TIM-3, while trophoblast cells show relatively low levels of expression. Additionally, hormone-induced decidualization significantly upregulates Galectin-9 expression in endometrial stromal cells. DISCUSSION: The research results suggest that Galectin-9 and TIM-3, as important immune co-signaling molecules, may play a crucial role in maintaining the immune-tolerant microenvironment at the maternal-fetal interface. Additionally, the association between decidualization and Galectin-9 expression reveals its potential role in pregnancy maintenance, providing new insights for the study of adverse pregnancy outcomes.