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Successful islet transplantation is dependent on the quality and quantity of islets infused. Islets are purified on density gradients, but procedures currently used have limited capacity for pancreatic digests, islet yield, and viability. We aimed to improve islet purification with a modified gradient medium. Biocoll was diluted in University of Wisconsin solution to create linear density gradients of 1.065 to 1.095 g/mL. Properties of islets purified from 22 human pancreas digests with modified medium were compared with 15 preparations using standard medium. The modification increased the capacity of gradients for pancreatic digests from 20 to 60 mL, islet yield increased from 218,000 to 435,318 per isolation, and viability increased from 65.4% to 92.1%. Islet fractions contained greater than 95% of recovered insulin. Islets showed good physiologic responses to secretagogues and restored normoglycemia in streptozotocin-induced diabetic severe combined immunodeficiency disease mice. The new medium enhances yield, purity, and viability of human islet preparations for clinical islet transplantation.