The present study investigated whether melatonin influences the expression/oligomerization of amylin with endoplasmic reticulum (ER) stress in rat insulinoma INS-1E cells. No change in cell survival after exposure to thapsigargin- and tunicamycin-combined melatonin treatment or melatonin-only treatment was observed when compared with the normal control cells. With thapsigargin-only or combined tunicamycin-melatonin treatments, phosphorylation of extracellular signal-regulated kinase (ERK) was significantly increased compared with control and melatonin-only treatments. A significant increase was observed in the levels of ER stress markers, namely, phosphorylated inositol-requiring protein 1α (p-IRE1α), CCAAT enhancer binding proteins (C/EBP)-homologous protein, p-eukaryotic translation initiation factor 2α and cleaved caspase-12, in the thapsigargin-combined melatonin-treated cells as compared with the tunicamycin-combined or only melatonin treatment. The melatonin-only treatment resulted in increased levels of amylin expression/oligomerization in 15-25 kDa and insulin proteins, compared with the thapsigargin- and tunicamycin-combined melatonin treatments. Treatment with ER stress inhibitor 4-phenylbutyric acid (4-PBA) did not suppress amylin expression/oligomerization or insulin production with thapsigargin or tunicamycin treatment. Levels of cleaved caspase-12 were significantly decreased in the thapsigargin- or tunicamycin-4-PBA combination treatments. Therefore, whether melatonin regulates the amylin expression/oligomerization in thapsigargin- or tunicamycin-combined with Bafilomycin A1 (autophagy inhibitor) or MG132 (proteasome inhibitor) treatments were investigated. Amylin expression/oligomerization with melatonin treatment was significantly decreased in the thapsigargin- or tunicamycin-combined Bafilomycin A1 or MG132 treatments. Since these outcomes are involved in cell viability, they indicate that increased cell death leads to decreased amylin expression/oligomerization, however, the effects of melatonin treatment on amylin expression/oligomerization induce proliferation of pancreatic β cells and improve the cellular functions of pancreatic β cells.