Mutations of the HNF4A gene cause a form of maturity-onset diabetes of the young (MODY1) that is characterized by impairment of pancreatic β-cell function. HNF4α is a transcription factor belonging to the nuclear receptor superfamily (NR2A1), but its target genes in pancreatic β-cells are largely unknown. Here, we report that ankyrin repeat and sterile α motif domain containing 4b (Anks4b) is a target of HNF4α in pancreatic β-cells. Expression of Anks4b was decreased in both βHNF4α KO islets and HNF4α knockdown MIN6 β-cells, and HNF4α activated Anks4b promoter activity. Anks4b bound to glucose-regulated protein 78 (GRP78), a major endoplasmic reticulum (ER) chaperone protein, and overexpression of Anks4b enhanced the ER stress response and ER stress-associated apoptosis of MIN6 cells. Conversely, suppression of Anks4b reduced β-cell susceptibility to ER stress-induced apoptosis. These results indicate that Anks4b is a HNF4α target gene that regulates ER stress in β-cells by interacting with GRP78, thus suggesting that HNF4α is involved in maintenance of the ER.