Background: Insulin dysregulation (ID) in horses is characterized by insulin resistance and exaggerated insulin responses, yet the underlying endocrine mechanisms remain incompletely understood. In humans, α-cell dysfunction, characterized by exaggerated glucagon secretion, contributes to metabolic disease; however, the role of pancreatic α-cell function in equine ID has not previously been investigated. Objectives: To determine whether horses with moderate to severe ID exhibit α-cell dysfunction, assessed by fasting and postprandial glucagon concentrations and postprandial suppression of glucagon secretion, compared with horses with normal insulin regulation (NIR). Study Design: Cross-sectional experimental study. Methods: Insulin dysregulated horses (n = 19) and NIR horses (n = 19) underwent a 180-minute seven-sampled oral sugar test. Plasma glucose, insulin, and glucagon concentrations were measured at baseline and every 30-minute for 180 minutes postprandially. Fasting glucagon concentrations (Fasting GLUCAGON), total area under the curve for glucagon (AUC GLUCAGON) and postprandial suppression of glucagon secretion were used as primary outcomes. Results: Horses with ID exhibited higher geometric mean (95% confidence interval) values compared to NIR horses for the α-cell dysfunction indices Fasting GLUCAGON (10.3 (8.0 – 13.4) vs 4.1 (3.5 – 4.8) pmol/L, P < 0.0001) and AUC GLUCAGON (1351.5 (1016.1 – 1797.7) vs 624.7 (529.8 – 736.6) pmol/L x min, P < 0.0001). Geometric least squares mean (95% confidence interval) glucagon concentrations were higher (P = 0.01) at 180 minutes after oral sugar administration in ID horses (6.3 (5.0 – 8.0) pmol/L) compared with baseline concentrations in NIR horses (4.1 (3.3 – 5.2) pmol/L), indicating impaired postprandial glucagon suppression in the ID group. Main Limitations: Cross-sectional design and unmatched groups limit causal inference. Conclusions: Horses with moderate to severe ID exhibit marked α-cell dysfunction characterized by fasting and postprandial hyperglucagonemia and impaired postprandial suppression of glucagon secretion. These findings identify α-cell dysregulation as a novel component of equine ID.