In rat diabetic animal models treatment with angiotensin-(1-7) [Ang (1-7)] prevents the development of diabetic cardiovascular complications. Angiotensin-converting enzyme 2 (ACE2) is a major Ang (1-7) generating enzyme in vascular smooth muscle cells (VSMCs) and its expression is decreased by prolonged exposure to high glucose (HG). However, the underlying mechanism of its down-regulation is unknown and was the subject of this study. Rat aortic VSMCs were maintained in normal glucose (NG) or HG (~4.1 mmol/l and ~23.1 mmol/l, respectively) for up to 72 h. Several protein kinase C (PKC) and NADPH oxidase inhibitors and siRNA's were used to determine the mechanism of HG-induced ACE2 down-regulation. Cell lysates were subjected to SDS-PAGE and Western blot analysis, RTQ-PCR, and radioimmunodetection of Ang (1-7). At 72 h of HG exposure, ACE2 mRNA, protein, and Ang (1-7) levels were decreased (0.17±0.01-, 0.47±0.03-, and 0.16±0.01-fold, respectively), and expression of NADPH oxidase subunit Nox1 was increased (1.70±0.2-fold). HG-induced ACE2 decrease was reversed by NADPH oxidase inhibitors apocynin and DPI, and Nox1 siRNA, as well as by inhibitors of glycotoxins formation. ACE2 expression was PKCII-dependent and PKCII protein levels were reduced in the presence of HG (0.32±0.03-fold); however, PKCII inhibitor CG53353 prevented HG-induced ACE2 loss and Nox1 induction, suggesting nonspecific targeting of the inhibitor. Our data suggest that glycotoxins-induced Nox1 expression is regulated by conventional PKC. ACE2 expression is PKCII-dependent. Nox1-derived superoxides affect PKCII expression, which lower ACE2 mRNA and protein levels and consequently decreased Ang (1-7) formation.