Endothelial cell production of anticoagulant heparan sulfate (HS^act) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS^act dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS^act was evaluated by generating Hs3st1^–/– knockout mice. Hs3st1^–/– animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS^act but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS^act predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1^–/– and Hs3st1^+/+ mice yielded indistinguishable occlusion times and comparable levels of thrombin•antithrombin complexes. Thus, Hs3st1^–/– mice did not show an obvious procoagulant phenotype. Instead, Hs3st1^–/– mice exhibited genetic background–specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS^act. Surprisingly, this bulk of HS^act is not essential for normal hemostasis in mice. Instead, 3-OST-1–deficient mice exhibited unanticipated phenotypes suggesting that HS^act or additional 3-OST-1–derived structures may serve alternate biologic roles.