Intrinsic glomerular cells in a diabetic milieu have transcriptional activation of genes that influence the development of diabetic nephropathy. The cellular repertoire of microRNAs can regulate translation of these expressed genes into proteins. Fibronectin is a key matrix protein accumulated in excess in diabetic nephropathy. Here, we exposed cultured human and mouse mesangial cells to high glucose and transforming growth factor-beta to simulate the diabetic milieu. In these conditions in vitro, as well as in mouse diabetic nephropathy models in vivo, microRNA-377 was consistently up-regulated relative to controls. Through a combination of computational and biological approaches, we identified relevant miR-377 target genes. Although fibronectin was induced by miR-377, it was not a direct target of miR-377. However, miR-377 led to reduced expressions of p21-activated kinase and superoxide dismutase, which enhanced fibronectin protein production. Thus, overexpression of miR-377 in diabetic nephropathy indirectly leads to increased fibronectin protein production; as such, miR-377 can have a critical role in the pathophysiology of this prevalent human disease.