We surveyed reservoir animals of leptospires in the northern part of Miyazaki Prefecture, where a cluster of human leptospirosis had occurred during the summer of 2006. Leptospira was isolated from 6 of 57 large Japanese field mice (Apodemus speciosus). The serogroups of the isolates were Autumnalis (5 strains) and Hebdomadis (1 strain) and the partial nucleotide sequences of their flaB genes suggested that the isolates belonged to L. interrogans. The human patient sera reacted specifically with the Leptospira strain isolated from the mice captured around the area where each patient occurred, suggesting that mice are the source of human infection. We also detected leptospiral DNAs by flaB-polymerase chain reaction in the kidneys of large feral animals; wild boars (positive ratio 10.3%; 4 of 39) and deer (19.2%; 10 of 52). The Leptospira spp. harbored by these animals were deduced to be L. interrogans (in 5 animals) and L. borgpetersenii (in 9 animals) by the nucleotide sequences of the amplicons. Anti-Leptospira antibodies were also detected among symptomatic hound dogs. These results suggest that these feral animals may cause leptospirosis and pose a potential risk to hunters and workers in the meat processing industry.