The current available insulin therapies decrease blood glucose but are associated with the risk of developing hypoglycemia. Glucagon is a counter regulatory hormone and we hypothesize that a fixed ratio of insulin and a long-acting glucagon-analogue can reduce the risk of hypoglycemia. To define an appropriate ratio we tested two fixed glucagon doses (3.5 and 10 nmol/kg) in combination with increasing doses of insulin in diabetic rats. We observed a plateau in blood glucose at 15.2 mmol/L with 10 nmol/kg of the glucagon-analogue. The mechanism behind this plateau, protecting against hypoglycemia, was investigated by measuring the glucose output, cAMP production, and hormone binding in primary rat hepatocytes. While glucose output could contribute to the observed plateau in blood glucose, cAMP response or hormone binding did not explain the observation. Though such plateau indicated decreased risk of hypoglycemia a full normalization of blood glucose was still needed. Based on the data obtained with 3.5 nmol/kg of the glucagon-analogue, a 1:23 (glucagon-analogue:insulin) ratio was chosen and a dose-response was performed in diabetic rats. At low doses (<= 20 nmol/kg), insulin and the 1:23 ratio showed similar efficacy of lowering blood glucose. Interestingly, the insulin-dose resulting in hypoglycemia was increased from 40 nmol/kg insulin alone to 160 nmol/kg insulin in the 1:23 ratio. Analysis of the liver glycogen content at the end of the experiment showed that the highest dose in the 1:23 ratio almost emptied the liver from glycogen. Thus, liver glycogen is essential for the protective effect of glucagon in hypoglycemia.