Diabetic nephropathy, a major complication of diabetes, leads to kidney failure. Glucagon, a hormone that raises blood sugar, becomes abnormally elevated in diabetes due to insulin resistance, which can worsen hyperglycaemia and its complications. This study aims to investigate whether glucagon causes vasodilation in rat segmental arteries, potentially contributing to kidney damage, by using a myography system. Arteries were excised from a rat kidney, normalized, and activated with a wake-up protocol using Potassium Physiological Saline Solution and norepinephrine. Arteries were tested with acetylcholine to assess endothelial function. Glucagon was then added in increasing doses, followed by a final Potassium Physiological Saline Solution wash to confirm arterial responsiveness. Renal arteries remained functional throughout the experiment, as shown by the vasoconstriction elicited by the Potassium Physiological Saline Solution-induced contraction at the end of the protocol. Acetylcholine caused dose-dependent relaxation via muscarinic receptor activation, triggered nitric oxide release, and confirmed an intact and responsive endothelium. Glucagon also induced dose-dependent relaxation by activation of the glucagon receptor, which suggested a vasodilatory effect on renal arteries. This effect led to an increase in glomerular damage and a decrease in glomerular filtration rate, which contributed to kidney damage. New therapies targeting glucagon could slow kidney damage, improve outcomes, and reduce dialysis needs. Future research should explore glucagon’s signaling pathways and test effects in other vessels and disease models.