Increasing attention has been paid to the maternally inherited microbes that are capable of manipulating the reproduction of their hosts for their own benefit. Although several studies have revealed that the host genotype can affect the intensity of the manipulation, the underlying genetic basis is poorly understood. Here, we examined the intensity of spiroplasma-induced male killing in various wild-type stocks of Drosophila melanogaster to clarify the genetic basis of the host factors responsible for the variation in the male-killing intensity. Among ten lines examined by mating experiments (that is, nuclear introgression), eight lines including Oregon-R and Canton-S were found to have nuclear factors that allowed strong expression of male killing. In contrast, the nuclear factors of the lines Sevelen and Hikone partially suppressed or remarkably retarded the expression of male killing. These results were confirmed by artificial transfer experiments of spiroplasma infection across the fly lines by means of microinjection. A series of mating experiments revealed that the nuclear factors acting against male killing were mainly located on autosomes in Sevelen and on the X chromosome in Hikone. In both lines, the suppressors were inferred to act maternally with a dominant effect. The nuclear factors of Sevelen and Hikone scarcely affected spiroplasma densities in reproductively active young insects, suggesting that the suppressors may act on the male-killing expression directly rather than through suppressing bacterial proliferation.