In butterflies, the adult sex ratio observed in the field is usually male-biased, although the sex ratio of their progeny is 1:1. This is due to the higher motility and larger behavioral range of males than females. As expected, the sex ratio of Eurema hecabe butterflies collected at 6 localities throughout Japan was male-biased. However, in Tsukuba, located in the central part of Japan, the sex ratio was found to be biased toward females. Their progeny reared in the laboratory also exhibited a female-biased sex ratio. A single strain of Wolbachia is considered to be the cause of the sex-ratio distortion, because antibiotic treatment reversed the sex ratio to 1:1, and only a single nucleotide sequence of wsp, a highly variable Wolbachia gene, was detected by molecular analysis. Cytogenetic analysis excluded the possibility of feminization as the underlying mechanism. In addition, when the wild-caught females that had already mated in nature were treated with antibiotics before oviposition, egg-hatch rates were extremely low, suggesting that the same Wolbachia strain also caused cytoplasmic incompatibility. Our findings suggest the possibility that a single strain of Wolbachia induces 2 distinct reproductive manipulations in the same host.