We isolated Acanthogobius hasta mitochondrial DNA by long-polymerase chain reaction (long-PCR) with conserved primers, and sequenced this mitogenome with primer walking. The resultant A. hasta mitochondrial DNA sequence was found to consist of 16,663 bp with a structural organization conserved relative to that of other fish. In this paper, we report the basic characteristics of the A. hasta mitochondrial genome including structural organization, base composition of rRNAs and the tRNAs and protein-encoding genes, and characteristics of mitochondrial tRNAs. These findings are applicable to molecular phylogenetics in the suborder Gobioidei.