Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the transforming growth factor-β superfamily, and is highly expressed in platelets and endothelial cells. We previously demonstrated its positive role in thrombus formation using a zebrafish thrombosis model. In the present study, we used Bambi-deficient mice and radiation chimeras to evaluate the function of this receptor in the regulation of both hemostasis and thrombosis. We show that Bambi−/− and Bambi+/− mice exhibit mildly prolonged bleeding times compared with Bambi+/+ littermates. In addition, using 2 in vivo thrombosis models in mesenterium or cremaster muscle arterioles, we demonstrate that Bambi-deficient mice form unstable thrombi compared with Bambi+/+ mice. No defects in thrombin generation in Bambi−/− mouse plasma could be detected ex vivo. Moreover, the absence of BAMBI had no effect on platelet counts, platelet activation, aggregation, or platelet procoagulant function. Similar to Bambi−/− mice, Bambi−/− transplanted with Bambi+/+ bone marrow formed unstable thrombi in the laser-induced thrombosis model that receded more rapidly than thrombi that formed in Bambi+/+ mice receiving Bambi−/− bone marrow transplants. Taken together, these results provide strong evidence for an important role of endothelium rather than platelet BAMBI as a positive regulator of both thrombus formation and stability.