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ObjectivesTo evaluate the efficacy of two different formulations of glucagon given by three different routes (subcutaneous; SQ, rectal, intranasal; IN) for correcting insulin-induced hypoglycaemia in cats and to describe any adverse effects and compare the time required for administration by each route.MethodsA randomized, nonblinded, sham-controlled crossover study was conducted in six healthy cats, with a 7-day washout period between treatments. Hypoglycemia was induced using a continuous rate infusion (CRI) of rapid-acting Lispro insulin. Glucagon nasal powder (single-dose, ready-to-use) was given intranasally and rectally and compared to subcutaneous glucagon injection and intranasal sham control. Blood glucose concentration (BG) was measured from samples collected at baseline (T-15), after induction of hypoglycemia (T0), and at 5-minute intervals for 30 minutes (min), and then at 15 min intervals for 1 hour (h) following glucagon administration. Plasma glucagon and potassium (K+) concentrations were measured at T-15, T0, and T15 and T30 following glucagon administration. The time taken to prepare and administer each treatment was recorded.ResultsMedian BG was 2.6 mmol/L at T0 and increased significantly following glucagon administration by all routes, reaching peak median concentrations of 11.2 mmol/L (intranasal), 9.7 mmol/L (rectal), and 10.15 mmol/L (subcutaneous). Plasma glucagon concentrations increased markedly by T15, with the highest concentrations observed following intranasal administration (1562 pmol/L), followed by subcutaneous (191.7 pmol/L) and rectal (158 pmol/L) routes of administration. Plasma potassium decreased following insulin administration and increased modestly by T30 following glucagon administration by all routes. No serious adverse effects occurred following glucagon administration.Conclusions and Clinical ImportanceTransmucosal glucagon effectively restores BG towards baseline following insulin-induced hypoglycemia with minimal side effects. The IN route resulted in the highest plasma glucagon and BG concentrations compared to the rectal and subcutaneous routes of administration. Further studies are needed to quantify the efficacy and safety of transmucosal glucagon in diabetic cats in response to spontaneous insulin-induced hypoglycemia.