Gremlin is a bone morphogenic protein (BMP) antagonist and is elevated in diabetic kidney tissues. In the early course of diabetic nephropathy (DN), podocyte are injured. We studied the protein and gene expression of gremlin in mice podocytes cultured in hyperglycemia ambient. The role of gremlin on podocyte injury and the likely signaling pathways involved were determined. Expression of gremlin was visualized by confocal microscopy. Recombinant mouse gremlin and small interfering RNA (siRNA) targeting to gremlin1 identified the role played by gremlin on podocytes. Study of canonical (smad2/3) and non-canonical (p38MAPK and JNK1/2) transforming growth factor beta (TGFβ)/smad mediated signaling revealed the putative signaling mechanisms involved. Smad2/3 siRNA and TGFβ receptor inhibition (SB431542) were used to probe canonical TGFβ/smad signaling in gremlin-induced podocyte injury. Apoptosis of podocytes was measured by TUNEL assay. Gremlin expression was enhanced in high glucose cultured mouse podocytes, and was localized predominantly in the cytoplasm and negligibly on the cell membrane. Not only expression of nephrin and synaptopodin were decreased on treatment with gremlin, but also synaptopodin rearrangement and nephrin relocalization were evident. Knockdown gremlin1 or smad2/3 by siRNA, and inhibition of TGFβR (SB431542) attenuated podocyte injury. Inhibition of canonical TGF-β signal blocked the injury of gremlin on podocytes. In conclusion, gremlin was clearly elevated in high glucose cultured mouse podocytes, and likely employed endogenous canonical TGFβ1/Smad signaling to induce podocyte injury. Knockdown gremlin1 by siRNA may be clinically useful in the attenuation of podocyte injury. J. Cell. Biochem. 114: 2101–2113, 2013. © 2013 Wiley Periodicals, Inc.