Experimental lipotoxicity constitutes a model for β-cell demise induced by metabolic stress in obesity and type 2 diabetes. Fatty acid excess induces endoplasmic reticulum (ER) stress, which is accompanied by ER morphological changes whose mechanisms and relevance are unknown. We found that the GTPase dynamin-related protein 1 (DRP1), a key regulator of mitochondrial fission, is an ER resident regulating ER morphology in stressed β-cells. Inhibition of DRP1 activity using a GTP hydrolysis-defective mutant (Ad-K38A) attenuated fatty acid-induced ER expansion and mitochondrial fission. Strikingly, stimulating the key energy-sensor AMP-activated protein kinase (AMPK) increased the phosphorylation at the anti-fission site Serine 637 and largely prevented the alterations in ER and mitochondrial morphology. Expression of a DRP1 mutant resistant to phosphorylation at this position partially prevented the recovery of ER and mitochondrial morphology by AMPK. Fatty acid-induced ER enlargement was associated with proinsulin retention in the ER, together with increased proinsulin/insulin ratio. Stimulation of AMPK prevented these alterations, as well as mitochondrial fragmentation and apoptosis. In summary, DRP1 regulation by AMPK delineates a novel pathway controlling ER and mitochondrial morphology, thereby modulating the response of β-cells to metabolic stress. DRP1 may thus function as a node integrating signals from stress regulators, such as AMPK, to coordinate organelle shape and function.