Aim/hypothesis. The objective of this study was to assess how long-term exposure to high glucose affects the a cell function and whether the increased glucagon secretion is mediated via insulin resistance. Materials and methods. We established a beta cell-depleted rat model to obtain pure primary alpha cells. Furthermore, isolated rat islets and TC1-6 cells (a clonal alpha cell line) were exposed to high glucose (25 or 30 mmol/L) and low glucose (5.5 mmol/L) for up to 5 days to evaluate the influence of chronic glucose toxicity on glucagon secretion and glucagon gene expression. Moreover, we added insulin and/or Wortmannin to examine if the inhibitory effect of insulin on glucagon secretion was impaired by high glucose via the phosphatidylinositol 3 kinase/PKB protein kinase B pathway. Results. Both glucagon secretion and glucagon gene expression were increased in response to 5 days exposure to high glucose. While a moderate insulin concentration slightly inhibits glucagon secretion from rat islets and alpha TC1-6 cells at high glucose, a pronounced increase in glucagon secretion was observed at low glucose. We found that the insulin-mediated activity of the phosphatidylinositol 3 kinase/PKB protein kinase B pathway in the alpha cell was markedly impaired by chronic exposure to high glucose. Conclusion. The hypersecretion of glucagon induced by glucotoxicity may be secondary to insulin resistance of the alpha cell induced by impaired activity of the insulin signaling pathway.