During the development of type 2 diabetes, endoplasmic reticulum (ER) stress leads to pancreatic beta cell failure. CCAAT/enhancer-binding protein (C/EBP) beta is highly induced by ER stress and AMP-activated protein kinase (AMPK) suppression in pancreatic beta cells, and its accumulation reduces pancreatic beta cell mass. We investigated the phosphorylation state of C/EBP beta under these conditions. Casein kinase 2 (CK2) was found to co-localize with C/EBP beta in MINE cells. It phosphorylated S222 of C/EBP beta, a previously unidentified phosphorylation site. We found that C/EBP beta is phosphorylated by CK2 under AMPK suppression and ER stress, which are important from the viewpoint of the worsening pathological condition of type 2 diabetes, such as decreased insulin secretion and apoptosis of pancreatic beta cells. (C) 2018 Elsevier Inc. All rights reserved.