Background: Diabetic kidney disease (DKD) is the leading cause of kidney failure in North America. DKD is characterized by low glomerular filtration. High glucose (HG) promotes the production of ECM protein in glomerular mesangial cell (MC) through the cytokines TGFβ1 and activins. We recently showed that TGFβ1 upregulation of microRNA (miR) 299a-5p promoted its profibrotic responses in MC. Here we studied the role of this miR in DKD. Methods: Primary mouse MC were treated with HG at 30 mM. miR299a-5p was detected by qPCR or ISH. miR overexpression and inhibition plasmids were transfected by electroporation. TGFβ1 and activin signaling was assessed by activity of their downstream mediator Smad3. ECM production was assessed. Results: HG increased the expression of miR299a-5p in MC. This was also validated in type 1 diabetic Akita kidneys in both glomeruli and tubules by ISH. In MC, miR299a-5p overexpression increased Smad3 activation and COL1α1 promoter activity. Conversely, miR299a-5p inhibition attenuated HG-induced COL1α1 promoter and Smad3 activation, as well as upregulation of ECM proteins. miR299a-5p is predicted to target the antifibrotic proteins Cripto-1 (CR-1), and follistatin (FST). Here we show that HG decreased protein expression of both CR-1 and FST was mediated through miR299a-5p. Conclusion: These data support an important role for miR299a-5p in regulation of the profibrotic response to HG. Through suppression of two important antifibrotic proteins, CR-1 and FST, miR299a-5p potentiates the action of TGFβ family profibrotic cytokines.