Glucosamine is a popular nutritional supplement used to treat osteoarthritis. Intravenous administration of glucosamine causes insulin resistance and endothelial dysfunction. However, rigorous clinical studies evaluating the safety of oral glucosamine with respect to metabolic and cardiovascular pathophysiology are lacking. Therefore, we conducted a randomized, placebo-controlled, double-blind, crossover trial of oral glucosamine at standard doses (500 mg p.o. t.i.d.) in lean (n = 20) and obese (n = 20) subjects. Glucosamine or placebo treatment for 6 weeks was followed by a 1-week washout and crossover to the other arm. At baseline, and after each treatment period, insulin sensitivity was assessed by hyperinsulinemic-isoglycemic glucose clamp (SI(Clamp)) and endothelial function evaluated by brachial artery blood flow (BAF; Doppler ultrasound) and forearm skeletal muscle microvascular recruitment (ultrasound with microbubble contrast) before and during steady-state hyperinsulinemia. Plasma glucosamine pharmacokinetics after oral dosing were determined in each subject using a high-performance liquid chromatography method. As expected, at baseline, obese subjects had insulin resistance and endothelial dysfunction when compared with lean subjects (SI(Clamp) [median {25th-75th percentile}] = 4.3 [2.9-5.3] vs. 7.3 [5.7-11.3], P < 0.0001; insulin-stimulated changes in BAF [% over basal] = 12 [-6 to 84] vs. 39 [2-108], P < 0.04). When compared with placebo, glucosamine did not cause insulin resistance or endothelial dysfunction in lean subjects or significantly worsen these findings in obese subjects. The half-life of plasma glucosamine after oral dosing was approximately 150 min, with no significant changes in steady-state glucosamine levels detectable after 6 weeks of therapy. We conclude that oral glucosamine at standard doses for 6 weeks does not cause or significantly worsen insulin resistance or endothelial dysfunction in lean or obese subjects.