ABSTRACTCoxsackievirus is an enteric virus that initiates infection in the gastrointestinal tract before disseminating to peripheral tissues to cause disease, but intestinal factors that influence viral replication are understudied. Furthermore, a sex bias for severe sequelae from coxsackievirus infections has been observed in humans. While mouse models mimicking human pathogenesis have been well characterized, many of these experiments use intraperitoneal injection of coxsackievirus to infect mice, bypassing the intestine. In light of recent studies identifying intestinal factors, such as the microbiota, that alter enteric viral replication, we sought to investigate coxsackievirus replication within the intestine. Here, we orally infected mice with coxsackievirus B3 (CVB3) and found that CVB3 replication in the intestine is sex dependent. CVB3 replicated efficiently in the intestine of male mice but not female mice. Additionally, we found that the type I interferon response and sex hormones can alter both viral replication and lethality. Overall, these data suggest that sex and the immune response play a vital role in CVB3 replication in the intestine and should be considered in light of the sex bias observed in human disease.IMPORTANCESex bias in severe sequelae from enteric viral infections has been observed. Since viruses have evolved to achieve optimal levels of fitness in their environmental niches, it is imperative to study viruses at the site of initial replication. Here, we used an oral inoculation system for CVB3, which follows the natural route of infection in the gastrointestinal tract. We found that sex can influence the replication of CVB3 in the intestine. Additionally, the type I interferon response and sex hormones alter both CVB3 intestinal replication and lethality. Overall this work highlights the fact that sex should be considered in investigations of enteric viral replication and pathogenesis.