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Background: Although most biologics are produced using recombinant technologies, heparin persists as a product purified from animal tissues. A cell based system for production of heparin would eliminate risk of supply shortage and contamination. Additionally, genetic engineering could yield heparin with improved qualities such as reduced risk of heparin-induced thrombocytopenia. Aims: This work is focused on engineering mammalian cell lines and bioprocess methods to produce recombinant heparin. Methods: The heparan sulfate biosynthetic pathway of mastocytoma cells was genetically engineered to alter the expression of heparan sulfate sulfotransferases. The resulting cell lines were screened for production of anti-FXa activity. Heparan sulfate production from a candidate cell line was tested in chemically defined medium. The recombinant product was characterized structurally and in clotting, anti-protease and heparin induced thrombocytopenia assays. Results: Engineered cells produced heparan sulfate in chemically defined medium with anti-Xa and anti-IIa activity exceeding the requirement for unfractionated heparin despite having lower sulfate content. Chain length was longer than unfractionated heparin. Additionally, binding to platelet factor 4 was reduced compared to unfractionated heparin, suggesting less risk of heparin-induced thrombocytopenia. Conclusion: These results demonstrate the feasibility of producing a substitute for unfractionated heparin from recombinant cell culture. Additionally, recombinant technology may allow production of heparin substitutes with improved properties such as reduced side effects.