Autophagy proceeds through several defined steps, with autophagosome formation being critical for the delivery of cytoplasmic components to the lysosome or vacuole for degradation. The initiation of autophagosome formation requires the recruitment of a complex that consists of FIP200–ATG13–ULK1 to the endoplasmic reticulum (ER), but it remains unclear how this recruitment occurs. Calcium (Ca2+) chelation blocks autophagosome formation, which suggests Ca2+ as a potential mediator. Using multi-modal structured illumination microscopy combined with a sensitive Ca2+ sensor, Zheng et al. detected Ca2+ oscillations on the ER cytosolic surface in response to autophagy induction. After stimulation of Ca2+ transients, FIP200 underwent a phase separation from a diffuse pattern to ER-adjacent fusion-prone liquid-like FIP200 condensates. These condensates primed ER tubules as the site of autophagosome generation through interaction with the ULK1 complex. EPG-4 (EI24), a metazoan-specific ER autophagy protein, was identified as regulating the ER Ca2+ oscillations and required attachment of FIP200 to the ER to function. Deletion of EI24 led to increased frequency and amplitude of ER Ca2+ transients and autophagy defects. This study provides another example of phase separation as a fundamental mechanism for regulating key biological processes.