Blockade of the glucagon receptor (GCGR) has been shown to improve glycemic control. However, this therapeutic approach also brings side effects, such as α-cell hyperplasia and hyperglucagonemia, and the mechanisms underlying these side effects remain elusive. Here, we conduct single-cell transcriptomic sequencing of islets from male GCGR knockout (GCGR-KO) mice. Our analysis confirms the elevated expression of Gcg in GCGR-KO mice, along with enhanced glucagon secretion at single-cell level. Notably, Vgf (nerve growth factor inducible) is specifically upregulated in α cells of GCGR-KO mice. Inhibition of VGF impairs the formation of glucagon immature secretory granules and compromises glucagon maturation, lead to reduced α-cell hypersecretion of glucagon. We further demonstrate that activation of both mTOR-STAT3 and ERK-CREB pathways, induced by elevated circulation amino acids, is responsible for upregulation of Vgf and Gcg expression following glucagon receptor blockade. Thus, our findings elucidate parts of the molecular mechanism underlying hyperglucagonemia in GCGR blockade.