Sulfonylurea and metformin are used in the treatment of diabetes. Their chronic effects on β cells are not well known. We have shown that sustained exposure of rat β cells to glibenclamide increased their protein synthesis activity, while metformin caused an inhibition. The effect of glibenclamide was attributed to an activation of translation factors. This study examines whether both drugs interact at the level of protein translation in β cells. Purified rat β cells were cultured with and without glibenclamide and metformin before measurement of protein and insulin synthesis, abundance of (phosphorylated) translation factors, and cell viability. A 24 h exposure to metformin stimulated AMP-activated protein kinase (AMPK), suppressed activation of translation factors- both the mammalian target of rapamycin (mTOR; also known as mechanistic target of rapamycin, MTOR)-dependent ones (eukaryotic initiation factor 4E-binding protein 1 and ribosomal protein S6) and the mTOR-independent eukaryotic elongation factor 2-, and inhibited protein synthesis; a 72 h exposure resulted in 50% dead cells. These effects were counteracted by addition of glibenclamide, the action of which was blocked by the mTOR inhibitor rapamycin and the protein kinase A (PKA) inhibitor Rp-8-Br-cAMPs. In conclusion, metformin activates AMPK in β cells leading to suppression of protein translation through mTOR-dependent and -independent signaling. Glibenclamide antagonizes these metformin effects through activation of mTOR- and PKA-dependent signaling pathways.