To determine whether deer can transmit Neospora caninum, brains of naturally infected white-tailed deer (Odocoileus virginianus) were fed to 4 dogs; 2 of these dogs shed oocysts. Oocysts from 1 of the dogs were tested by polymerase chain reaction and found to be positive for N. caninum and negative for Hammondia heydorni. The internal transcribed spacer 1 sequence of the new strain (designated NC-deer1) was identical to N. caninum from domestic animals, indicating that N. caninum is transmitted between wild and domestic animals, often enough to prevent divergent evolution of isolated populations of the parasite. NC-deer1 oocysts were administered to a calf that developed a high antibody titer, providing evidence that N. caninum from wildlife can infect cattle. In addition, N. caninum antibody seroprevalence was detected in 64/164 (39%) free-ranging gray wolves (Canis lupus), 12/113 (11%) coyotes (Canis latrans), 50/193 (26%) white-tailed deer, and 8/61 (13%) moose (Alces alces). These data are consistent with a sylvatic transmission cycle of N. caninum between cervids and canids. We speculate that hunting by humans favors the transmission of N. caninum from deer to canids, because deer carcasses are usually eviscerated in the field. Infection of canids in turn increases the risk of transmitting the parasite to domestic livestock.