The relationship between metabolic syndrome and vascular dysfunction is a prominent factor in the development of cardiovascular diseases. Previous data from our laboratory demonstrated functional and structural abnormalities within the aortic vasculature of the high-fat diet (HFD)-streptozotocin (STZ)-induced diabetes mellitus (HFD-D) rat model of metabolic syndrome. We further showed that a direct association between altered metabolic profile and deficits in endothelium-dependent relaxation exists. In the present study, changes in nitric oxide (NO) pathway, a critical determinant of endothelium-dependent relaxation, and their association with impaired metabolic parameters in HFD-D rats was investigated. Five-week-old male Wistar albino rats (n = 24) were fed with either HFD (45 kcal% fat) or control diet (10 kcal% fat) for 10 weeks. On week 6, 40 mg/kg STZ and saline were injected (i.p.) into the HFD and control groups, respectively. By week 10, tail systolic blood pressure (SBP) was recorded, and metabolic data were collected. Subsequently, the rats were euthanized, and blood, liver, and kidney samples were collected. Maximal NO-dependent vasorelaxation was assessed in abdominal aortic rings precontracted with phenylephrine (1 µM) and preincubated with the nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 10 µM). Collected measures also included fasting blood glucose (FBG), plasma total cholesterol (TC), plasma triglyceride (TG), plasma high-density lipoprotein (HDL), plasma creatinine (Cr), urinary protein: urinary creatinine (UPC), liver index (LI), and kidney index (KI). HFD-D rats had higher SBP, FBG, TG, Cr, UPC, LI and KI, lower HDL and unchanged TC compared with controls. Maximal NO-induced vasorelaxation (NO Rmax) was markedly blunted in the HFD-D rats compared with controls. NO Rmax showed negative correlations with FBG, TG, Cr, UPC, LI, and KI, while positive correlations were observed with HDL and TC. No significant correlation was found between NO Rmax and SBP. Our previous demonstration of impaired endothelial-dependent relaxation in metabolic syndrome is likely contributed to by a parallel impairment in NO-induced vasorelaxation. It is also plausible that deficits in NO-dependent vasorelaxation stem from the disturbed metabolic function observed in this model. Further research is warranted to provide a comprehensive mechanistic explanation to the above findings.