Most animal cells rely on aerobic metabolism for survival and are damaged or die within minutes without oxygen. Embryos of the annual killifish Austrofundulus limnaeus, however, survive months without oxygen. Determining how their cells survive without oxygen has the potential to revolutionize our understanding of the cellular mechanisms supporting vertebrate anoxia tolerance and the evolution of such tolerance. Therefore, we aimed to establish and characterize an anoxia-tolerant cell line from A. limnaeus for investigating mechanisms of vertebrate anoxia tolerance. The PSU-AL-WS40NE cell line of neuroepithelial identity was established from embryonic tissue of A. limnaeus using a tissue explant. The cells can survive for at least 49 d without oxygen or replenishment of growth medium, compared to only 3 d of anoxic survival for two mammalian cell lines. PSU-AL-WS40NE cells accumulate lactate during anoxia, indicating use of common metabolic pathways for anaerobic metabolism. Additionally, they express many of the same small noncoding RNAs that are stress-responsive in whole embryos of A. limnaeus and mammalian cells, as well as anoxia-responsive small noncoding RNAs derived from the mitochondrial genome (mitosRNAs). The establishment of the cell line provides a unique tool for investigating cellular mechanisms of vertebrate anoxia tolerance, and has the potential to transform our understanding of the role of oxidative metabolism in cell biology.