Mitochondrial gene content shows extensive variation among eukaryotes, but is remarkably compact and static in bilateral animals. Mitochondrial genomes of bilaterians typically contain two rRNA, 22 tRNA, and 13 protein-coding genes. In this study, we report that the mitochondrial genomes of Antarctic fishes of the suborder Notothenioidei (Perciformes) lack two adjacent genes encoding NADH 6 dehydrogenase (ND6) and tRNA(Glu). Loss of the ND6 gene is reported for the first time in an animal mitochondrial genome, and is considered an extremely rare evolutionary event. Dot blot and ND6 transcript detection analyses found no evidence of mitochondrial ND6 gene copies in heteroplasmy or of a functional ND6 gene copy in the nuclear genome, respectively. Hence, we concluded that ND6 function was lost in Antarctic notothenioids, and could be compensated for by functional changes in other proteins of the mitochondrial respiratory system.