Diabetic cardiomyopathy (DCM) is one of the main causes of heart failure in patients with diabetes. Cardiac fibrosis caused by endothelial mesenchymal transformation (EndMT) plays an important role in the pathogenesis of DCM. NLRC5 is a recently discovered immune and inflammatory regulatory molecule in the NOD-like receptor family, and is involved in organ fibrosis. In this study, we found that the expression of NLRC5 was up-regulated in endothelial cells (ECs) and cardiac fibroblasts (CFs) in diabetes models both in vivo and in vitro. NLRC5 knockdown significantly inhibited high glucose-induced EndMT. In addition, NLRC5 deficiency inhibited the expression of phosphorylated Smad2/3 and the activation of EndMT-related transcription factors in ECs induced by high glucose. However, the effect of NLRC5 deficiency on CFs was not obvious. In summary, our results suggest that NLRC5 deficiency ameliorates cardiac fibrosis in DCM by inhibiting EndMT through Smad2/3 signaling pathway and related transcription factors. NLRC5 is likely to be a biomarker and therapeutic target of cardiac fibrosis in diabetic cardiomyopathy. (C) 2020 Elsevier Inc. All rights reserved.