Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)–anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.