Patients with diabetic foot ulceration and critical limb ischemia have a high risk of major amputation, especially if limbs can not be revascularized. Urokinase is effective in improving microcirculation in critical limb ischemia and might improve outcomes. There are no data on the efficacy and safety of urokinase treatment (survival free of major amputation, ulcer healing and the rate of minor and major bleeding).Therefore,we aimed to investigate the effect of urokinase treatment in a phase II clinical trial. We performed an open, prospective, non-controlled, multicenter phase II cohort study in 77 type-2 diabetic patients with critical limb ischemia and diabetic foot ulceration. Patients had no surgical or endovascular treatment option based on interdisciplinary consensus. Urokinase (1 Mio IU if plasma fibrinogen ≥2.5 g/l, 0.5 Mio IU if fibrinogen < 2.5 g/l) was administered for 21 days as an intravenous infusion over 30 minutes. Each patient was followed up for 12 months.Treatment for a median of 21 days resulted in 33% of patients being alive, having no major amputation and completely healed ulcers after 12 months.Total survival rate was 84.6%, amputation-free survival 69.2% and rate of major amputation 21.1%. Eighty-two percent of patients experienced at least once a complete ulcer healing within the course of study.Three serious adverse events were urokinase-related. Urokinase treatment in diabetic patients with critical limb ischemia appears to be effective, feasible and safe.Although this calls for a larger, randomized and controlled trial, the results are highly relevant for clinical practice to prevent these patients from receiving major amputation due to diabetic foot syndrome.