Despite advances in management of immunosuppression, graft rejection remains a significant clinical problem in solid organ transplantation. Non-invasive biomarkers of graft rejection can facilitate earlier diagnosis and treatment of acute rejection. The purpose of this study was to investigate the potential role of heparan sulfate as a novel biomarker for acute cellular rejection. Heparan sulfate is released from the extracellular matrix during T-cell infiltration of graft tissue via the action of the enzyme heparanase. In a murine heart transplant model, serum heparan sulfate is significantly elevated during rejection of cardiac allografts. Moreover, expression of the enzyme heparanase is significantly increased in activated T-cells. In human studies, plasma heparan sulfate is significantly elevated in kidney transplant recipients with biopsy-proven acute cellular rejection compared to healthy controls, recipients with stable graft function, and recipients without acute cellular rejection on biopsy. Taken together, these findings support further investigation of heparan sulfate as a novel biomarker of acute cellular rejection in solid organ transplantation.