Austrofundulus limnaeus is an extremophile vertebrate native to small temporary ponds of Venezuela. Embryos of A. limnaeus must survive variable and often extreme conditions, including long periods of anoxia. Neuroepithelial cells derived from these embryos, WS40NE cells, provide a unique tool to understand how the proteome changes in response to anoxic stress. Using label-free proteomics, 19604 peptides and 3487 proteins were quantified in normoxic, 4d anoxic, and 24 h recovery WS40NE cells. Of these, 2612 proteins (74.9%) were statistically significantly differentially expressed in at least one comparison: 1988 comparing normoxia to 4d anoxia (57.0%), 923 comparing 4d anoxia to 24 h recovery (26.5%), and 1814 comparing normoxia to 24 h recovery (52.0%). Further, interaction networks of proteins with similar expression patterns suggest that relative mitochondrial capacity may increase during anoxia, including upregulation and/or preferred stabilization of proteins involved in mitochondrial metabolism and mitochondrial transcription and translation. This is in sharp contrast to trends in proteins that support cytoplasmic translation. These data support an active role for mitochondria in mediating the survival of anoxia tolerant killifish cells.