Purpose <p>miR-138-5p plays a tumor-suppressing role, but its function in Wilms tumor (WT) has not yet been explored. This study aimed to investigate the role of miR-138-5p in the progression of WT.</p> Methods <p>Hematoxylin‒eosin staining was used to analyze histopathological changes in WT tissues and adjacent normal tissues. qPCR was used to detect the expression of miR-138-5p in WT tissues and cells. Immunohistochemical staining and qPCR were used to detect the expression of TCF3. The interaction between miR-138-5p and TCF3 was confirmed by correlation analysis, qPCR, and dual-luciferase assays, respectively. CCK-8, colony formation, Transwell, and wound-healing assays were performed to identify the effects of miR-138-5p and TCF3 on the proliferation and migration of WiT49 cells. A murine xenograft model was used to investigate the function of miR-138-5p on WT growth in vivo.</p> Results <p>miR-138-5p was expressed at low levels in WT tissues and WiT-49 cells. Functional studies demonstrated that the upregulation of miR-138-5p markedly inhibited the proliferation, colony-formation capacity, and migratory/invasive potential of WiT-49 cells. Subsequently, the therapeutic potential of miR-138-5p overexpression was confirmed in murine xenograft models, which markedly inhibited tumor growth compared with that in the control group. TCF3 is a target of miR-138-5p, and its expression is negatively correlated with that of miR-138-5p. Furthermore, the results of rescue experiments indicated that the tumor-suppressing effects of miR-138-5p in WiT-49 cells were reversed by TCF3 overexpression.</p> Conclusions <p>miR-138-5p could be a tumor suppressor that regulates the proliferation of WT cells by targeting and reducing TCF3 expression.</p>