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The aim of this study was to investigate the relationship between a sub-population of endothelial progenitor cells (EPC), namely colony-forming unit-endothelial cells (CFU-EC), their colony-forming capacity and variable clinical parameters, including insulin resistance and oxidative stress, in obese individuals. Thirty-eight obese adults (aged 42.5 ± 12.7), with BMI 32.3 ± 4.0 and 13 normal-weight controls (aged 48.2 ± 12.9; BMI 23.2 ± 2.3) were studied. CFU-EC colony-forming capacity was impaired in the group of obese individuals compared to the normal-weight controls (P = 0.001). The inverse correlation between homeostasis model assessment-insulin resistance (HOMAIR) index and CFU-EC number (r = −0.558, P < 0.0001) as well as positive total antioxidant status of plasma (TAS)/CFU-EC relation were noticed during the study. Additionally, correlations between the concentration of triglycerides (TG), high-density lipoproteins (HDLs), and body composition parameters in the obese participants were established. Our results demonstrate that insulin resistance and oxidative stress have a significant impact on the CFU-EC colony formation in obesity. Moreover, in multivariate regression analysis, in both studied groups, the HOMAIR index and HDL concentration were independent predictors of the number of CFU-EC. Endothelium dysfunction, which can be present in obesity, may in part be caused by EPC function impairment in this condition.