The male-killing symbiont Arsenophonus nasoniae is a gamma-proteobacterium that infects parasitic wasps; the male progeny of infected females exhibit increased embryonic death. In this study, we examined methods to horizontally infect Pteromalus venustus (a parasitoid infesting populations of the alfalfa leafcutting bee Megachile rotundata) with A. nasoniae. We then tested the success of these methods via semi-quantitative PCR and quantitative digital PCR, using a molecular marker specific to A. nasoniae. Controlled parasitoid mating experiments were then undertaken to determine whether infections of A. nasoniae in P. venustus induce the male-killing phenotype as has been reported for other host species; evidence of this male-killing phenotype was observed in the current study. Over the course of the eight parasitoid generations following introduction of A. nasoniae infection in P. venustus, the male component of the parasitoid sex ratio was substantially reduced in the infected population (1.05 ♂: 1.00 ♀) compared to the control population (2.46 ♂: 1.00 ♀). Establishment of stable A. nasoniae infections in P. venustus populations could lower the proportion of male progeny, thus negatively impacting the mating success of females, and reducing overall populations of the parasitoid in alfalfa leafcutting bee populations.