The significance of glucagon in the pathophysiology of diabetes mellitus is widely recognized, but the mechanisms underlying dysregulated glucagon secretion are still unclear. Here, we explored the molecular mechanisms of glucagon dysregulation, using an in vitro model. Hamster-derived glucagon-secreting InR1G cells were exposed to high glucose (25 mM) levels for 12 h before analyzing glucagon secretion and the activity of components involved in insulin signaling. High-glucose treatment induced increased glucagon secretion in InR1G cells, which represents a hallmark of diabetes mellitus. This treatment reduced the phosphorylation of Akt, indicating the deterioration of insulin signaling. Simultaneously, oxidative stress and JNK activity were shown to be increased. The inhibition of JNK signaling resulted in the amelioration of high-glucose level-induced glucagon secretion. Abnormally elevated glucagon secretion in diabetes can be reproduced by high-glucose treatment of InR1G cells, and the involvement of high glucose-oxidative stress-JNK-insulin signaling pathway axis has been demonstrated. These data elucidate, at least partly, the previously unclear mechanism of abnormal glucagon secretion, providing insights into a potential novel approach to diabetes treatment, targeting glucagon.