The Neptune whelk, Neptunea arthritica, is a sublittoral sea snail from Pacific waters that has been a food resource and is commercially important for the coastal fisheries in northern Japan. This species showed a severe decline during the 1970s and 1980s, possibly because of overfishing, imposex caused by tributyltin (TBT) pollution and parasite infection. In the present study, we investigated genetic variation among the populations of N. arthritica from eight localities in northern Japan, including Hokkaido and Aomori, using a mitochondrial DNA (mtDNA) marker, partial sequence of the cytochrome c oxidase subunit I (COI) gene, to compare the obtained results with those from previous microsatellite analyses. We also addressed the evolutionary history of N. arthritica and human impact on the population genetic profiles of this species. The parsimony network showed 14 COI haplotypes separated into 2 groups (Groups A and B), with an intermediate haplotype connecting both of the groups. Among eight populations, six were fixed for only one or two haplotypes, and any geographic-genetic correlation was not found; they were probably affected by random drift of the mtDNA lineage. Thus, the results from mtDNA contrasted with those from previous microsatellite analysis, indicating that geographic structure was affected by the restricted gene flow between populations. Our results suggested that N. arthritica diversified into Groups A and B during the Pliocene; however, recent TBT pollution and size-selective fishing pressure have reduced genetic diversity and concealed the natural population structure. The present study also suggested that human impact may cause long and possibly irreversible modification of ecosystems, particularly for species forming discrete and relatively small local populations, such as N. arthritica. Thus, the combined use of mtDNA and microsatellite genetic data provides a powerful tool to investigate the health of biodiversity in molluscs and shows that the output results of such analyses are of great interest for the conservation and management of molluscan species.