Podocytes are highly specialized cells that play an essential role in maintaining the integrity and function of the glomerular filtration barrier. Wilms tumor 1 (WT1) and β‐catenin are two master regulators that play opposing roles in podocyte biology and mutually antagonize each other. However, exactly how β‐catenin inhibits WT1 remains incompletely understood. In this study, we demonstrated the role of miR‐466o‐3p in mediating β‐catenin‐triggered podocyte injury by targeting WT1. The expression of miR‐466o‐3p was upregulated in cultured podocytes after β‐catenin activation and in glomerular podocytes in adriamycin (ADR) nephropathy, remnant kidney after 5/6 renal ablation, and diabetic kidney disease. Bioinformatics analysis and luciferase reporter assay confirmed that miR‐466o‐3p directly targeted WT1 mRNA. Furthermore, overexpression of miR‐466o‐3p downregulated WT1 protein and promoted podocyte injury in vitro. Conversely, inhibition of miR‐466o‐3p alleviated β‐catenin‐induced podocyte dysfunction. In mouse model of ADR nephropathy, overexpression of miR‐466o‐3p inhibited WT1, aggravated podocytes injury and deteriorated proteinuria. In contrast, inhibition of renal miR‐466o‐3p by antagomiR, either prior to or after ADR injection, substantially restored WT1, alleviated podocytes injury and reduced renal fibrosis. These studies reveal a critical role for miR‐466o‐3p, a novel microRNA that has not been characterized previously, in mediating β‐catenin‐triggered WT1 inhibition. Our findings also uncover a new pathogenic mechanism by which β‐catenin promotes podocyte injury and proteinuria in glomerular diseases.