Transmission experiments were performed to elucidate the life cycle of Sarcocystis zuoi found in Norway rats (Rattus norvegicus) in China. Two king rat snakes (Elaphe carinata) fed sarcocysts from the muscles of 4 naturally infected Norway rats shed sporocysts measuring 10.8 ± 0.7 × 8.0 ± 0.7 µm, with a prepatent period of 8–9 days. Sporocysts from the intestine of 2 experimentally infected king rat snakes were given to the laboratory Sprague–Dawley (SD) rats (R. norvegicus) and Kunming (KM) mice (Mus musculus). Microscopic sarcocysts developed in the skeletal muscles of SD rats. No sarcocysts were observed in KM mice. Characters of ultrastructure and molecule of sarcocysts from SD rats were confirmed as S. zuoi. Our results indicate that king rat snake is the definitive host of S. zuoi.