Enhanced Endoplasmic reticulum (ER) stress leads to cell death in various pathophysiological situations. During a search for compounds that regulate ER stress, we identified methoxyflavones, a group of flavonoids, as strong protective agents against ER stress-induced cell death. Analysis in mouse insulinoma MIN6 cells revealed that methoxyflavones mildly activated the eIF2 and Nrf2 pathways, but not the XBP1 pathway, and induced downstream genes including GRP78, a molecular chaperone in the ER. The protective effect of methoxyflavones was enhanced by agents that increase intracellular cAMP levels such as forskolin, dibutyryl cAMP (dbcAMP) and IBMX, but suppressed by a protein kinase A (PKA) inhibitor H-89, suggesting involvement of the PKA pathway in the regulation of ER stress by methoxyflavones. Consistent with the results in cultured cells, pretreatment of mice with tangeretin, a methoxyflavone, enhanced expression of GRP78 and HO-1 without causing ER stress in renal tubular epithelium, and prevented tunicamycin-induced cell death. Furthermore, pre-administration of tangeretin into mice enhanced expression of GRP78 in the substantia nigra pars compacta (SNpc), and protected dopaminergic neurons against MPTP, a neurotoxin that induces both oxidative and ER stress. These results suggest that methoxyflavones play an important role in the regulation of ER stress, and could be a therapeutic target for the ER stress-related diseases.