Low-carbohydrate diets (LCD) are increasing in popularity, but their effect on vascular health has been questioned. Endothelial microvesicles (EMV) are membrane-derived vesicles with the potential to act as a sensitive prognostic biomarker of vascular health and endothelial function. The aim of this study was to examine the influence of a LCD on EMV and other endothelial biomarkers of protein origin. Twenty-four overweight women (age, 48.4 ± 0.6 years; height, 1.60 ± 0.07 m; body mass, 76.5 ± 9.1 kg; body mass index, 28.1 ± 2.7 kg·m−2; waist circumference, 84.1 ± 7.4 cm; mean ± standard deviation) were randomised to either 24 weeks on their normal diet (ND) or a LCD, after which they crossed over to 24 weeks on the alternative diet. Participants were assisted in reducing carbohydrate intake, but not below 40 g·day−1. Body composition and endothelial biomarkers were assessed at the crossover point and at the end of the study. Daily carbohydrate intake (87 ± 7 versus 179 ± 11 g) and the percentage of energy derived from carbohydrate (29% versus 44%) were lower (p < 0.05) on the LCD compared to the ND, but absolute fat and saturated fat intake were unchanged. Body mass and waist circumference were 3.7 ± 0.8 kg and 3.5 ± 1.0 cm lower (p < 0.05), respectively, after the LCD compared with the ND phases. CD31+CD41−EMV, soluble (s) thrombomodulin, sE-selectin, sP-selectin, serum amyloid A and C-reactive protein were lower (p < 0.05) after the LCD compared to the ND, but serum lipids and apolipoproteins were not different. EMV along with a range of endothelial and inflammatory biomarkers are reduced by a LCD that involves modest weight loss.