Wolbachia are obligate intracellular bacteria that infect arthropods， including approximately two-thirds of insect species. Wolbachia manipulate insect reproduction by enhancing their inheritance through the female germline. The most common alteration is cytoplasmic incompatibility (CI)， where eggs from uninfected females fail to develop when fertilized by sperm from Wolbachia-infected males. By contrast， if female and male partners are both infected， embryos are viable. CI is a gene-drive mechanism impacting population structure and causing reproductive isolation， but its molecular mechanism has remained unknown. We show that a Wolbachia deubiquitylating enzyme (DUB) induces CI. The CI-inducing DUB， CidB， cleaves ubiquitin from substrates and is encoded in a two-gene operon， and the other protein， CidA， binds CidB. Binding is strongest between cognate partners in cidA-cidB homologues. In transgenic Drosophila， the cidA-cidB operon mimics CI when sperm introduce it into eggs， and a catalytically inactive DUB does not induce sterility. Toxicity is recapitulated in yeast by CidB alone; this requires DUB activity but is rescued by coexpressed CidA. A paralogous operon involves a putative nuclease (CinB) rather than a DUB. Analogous binding， toxicity and rescue in yeast were observed. These results identify a CI mechanism involving interacting proteins that are secreted into germline cells by Wolbachia， and suggest new methods for insect control.