AbstractProgressive albuminuria is the primary clinical symptom of diabetic nephropathy (DN), leading to a gradual decline in kidney function. DLX6-AS1 was the first reported long non-coding RNA (lncRNA) to participate in organogenesis and play crucial roles in the brain or neural cell development. Herein, we investigated the DLX6-AS1 (Dlx6-os1 in mice) role in DN pathogenesis. We found that DLX6-AS1 expression in DN patients correlated with the extent of albuminuria. Dlx6-os1 overexpression induced cellular damage and inflammatory responses in cultured podocytes through miR-346-mediated regulation of the GSK-3β pathway. In various established diabetic and newly developed knockout mouse models, Dlx6-os1 knockdown/knockout significantly reduced podocyte injury and albuminuria. The Dlx6-os1 effects were remarkably modulated by miR-346 mimics or mutants and significantly diminished in podocyte-specific GSK-3β-knockout mice. Thus, DLX6-AS1 (Dlx6-os1) promotes DN development by accelerating podocyte injury and inflammation through the upregulation of the GSK-3β pathway, providing a novel molecular target for DN therapy.