Almost any warm-blooded creature can be an intermediate host for Toxoplasma gondii. However, sexual reproduction of T. gondii occurs only in felids, wherein fertilisation of haploid macrogametes by haploid microgametes, results in diploid zygotes, around which a protective wall develops, forming unsporulated oocysts. Unsporulated oocysts are shed in the faeces of cats and meiosis gives rise to haploid sporozoites within the oocysts. These, now infectious, sporulated oocysts contaminate the environment as a source of infection for people and their livestock. RNA-Seq analysis of cat enteric stages of T. gondii uncovered genes expressed uniquely in microgametes and macrogametes. A CRISPR/Cas9 strategy was used to create a T. gondii strain that exhibits defective fertilisation, decreased fecundity and generates oocysts that fail to produce sporozoites. Inoculation of cats with this engineered parasite strain totally prevented oocyst excretion following infection with wild-type T. gondii, demonstrating that this mutant is an attenuated, live, transmission-blocking vaccine.