In this study, we investigated how melatonin mediates insulin synthesis through endoplasmic reticulum (ER) via HuD expression in rat insulinoma INS-1E cells. Under ER stress condition (thapsigargin with/without melatonin, tunicamycin with/without melatonin), phosphorylation of AMP-activated protein kinase (p-AMPK) was significantly increased when compared with only with/without melatonin (control/melatonin). Insulin receptor substrate (IRS) two protein was significantly reduced under conditions of ER stress when compared with control/melatonin, but no expression of IRS1 protein was observed. In thapsigargin treatment, melatonin (10, 50 μm) increased IRS2 protein expression in a dose-dependent manner. p-Akt (Ser473) expression significantly decreased under ER stress condition prior to control/melatonin. Melatonin (10, 50 μm) significantly reduced nuclear and cellular p85α expressions in a dose-dependent manner when compared with only thapsigargin or tunicamycin. These results indicate the activation of the aforementioned expressions under regulation of the pathway, AMPK → IRS2 → Akt/PKB → PI3K (p85α). However, mammalian target of rapamycin and raptor protein, mTORC1, was found to be independent of the ER stress response. In thapsigargin treatment, melatonin increased nuclear mammalian RNA-binding protein (HuD) expression and reduced cellular HuD expression and subsequently resulted in a decrease in cellular insulin level and rise in insulin secretion in a dose-dependent manner. In tunicamycin treatment, HuD and insulin proteins showed similar expression tendencies. These results indicate that ER stress/melatonin, especially thapsigargin/melatonin, increased nuclear HuD expression and subsequently resulted in a decrease in intracellular biosynthesis; it is hypothesized that extracellular secretion of insulin may be regulated by melatonin.