Background Increased levels of endothelial cell microparticles (EMP) are known to reflect endothelial dysfunction (ED). In diabetes mellitus type 2 (T2DM), the expression of endothelin (ET)‐1 is increased. As treatment with an ET‐1 antagonist significantly inhibited atherosclerosis in animal models, we sought to investigate whether treatment with ET‐1 antagonists affects EMP levels in vitro and in vivo in patients with T2DM. Materials and methods In vitro study: Human umbilical vein endothelial cells (HUVEC) were stimulated with ET‐1 alone and ET‐1 in combination with a dual ET‐A and ET‐B endothelin receptor blocker. In vivo study: Patients with T2DM were randomized to treatment with the ET receptor antagonist bosentan or placebo. After 4 weeks, the patients were re‐examined and blood samples were obtained. EMP counts in supernatants and plasma samples were determined using flow cytometry. Results In vitro study: In supernatants of ET‐1‐stimulated HUVECs, the increased release of EMP was reduced significantly by co‐incubation with an ET‐1 receptor antagonist (e.g. CD31+/CD42b‐EMP decreased from 37·1% ± 2·8 to 31·5% ± 2·8 SEM, P = 0·0078). In vivo study: No changes in EMP levels in blood samples of patients with T2DM were found after 4 weeks of bosentan treatment (n = 36, P = ns). Conclusions Our in vitro results suggest that ET‐1 stimulates the release of EMP from HUVECs via a receptor‐dependent mechanism. Co‐incubation with an endothelin receptor blocker abolished ET‐1‐dependent EMP release. However, treatment with bosentan for 4 weeks failed to alter EMP levels in patients with T2DM. Other factors seem to have influenced EMP release in patients with T2DM independent of ET‐1 receptor‐mediated mechanisms.