Autophagy is a cellular degradation process involving an intracellular membrane trafficking pathway that recycles cellular components or eliminates intracellular microbes in lysosomes. Many pathogens subvert autophagy to enhance their replication, but the mechanisms these pathogens use to initiate the autophagy process have not been elucidated. This study identifies rotavirus as a pathogen that encodes a viroporin, nonstructural protein 4, which releases endoplasmic reticulum calcium into the cytoplasm, thereby activating a calcium/calmodulin-dependent kinase kinase-β and 5' adenosine monophosphate-activated protein kinase-dependent signaling pathway to initiate autophagy. Rotavirus hijacks this membrane trafficking pathway to transport viral proteins from the endoplasmic reticulum to sites of viral replication to produce infectious virus. This process requires PI3K activity and autophagy-initiation proteins Atg3 and Atg5, and it is abrogated by chelating cytoplasmic calcium or inhibiting calcium/calmodulin-dependent kinase kinase-β. Although the early stages of autophagy are initiated, rotavirus infection also blocks autophagy maturation. These studies identify a unique mechanism of virus-mediated, calcium-activated signaling that initiates autophagy and hijacks this membrane trafficking pathway to transport viral proteins to sites of viral assembly.