Coxsackievirus B3 (CVB3) triggers viral myocarditis, with no effective vaccine yet. This fecal-orally transmitted pathogen has prompted interest in mucosal immunization to impede CVB3 spread. Our lab identified an attenuated strain, CVB3(mu), offering myocarditis and pancreatitis protection against CVB3(WT) in susceptible Balb/c mice. CVB3(mu)’s potential to stimulate mucosal immune defense remains to be elucidated. This study evaluates CVB3(mu)’s genetic stability via a rapid evolution cellular model and RNA sequencing. Its temperature sensitivity and safety were evaluated through in vitro and in vivo experiments. CVB3(mu)’s mucosal immunity protection was assessed via intranasal immunization in Balb/c mice. Results show CVB3(mu) maintains genetic stability and temperature sensitivity, retaining attenuated traits up to the 25th passage. Intranasal immunization elicited a significant Th1 response, potent serum neutralizing antibodies, and a substantial sIgA response in nasal washes. In vivo trials revealed CVB3(mu) protection in adult mice and passive protection in suckling mice against lethal CVB3(WT) challenges. In conclusion, CVB3(mu), a live attenuated intranasal vaccine, provides tripartite protection involving humoral, mucosal, and cellular immunity, making it a potential candidate to control CVB3 spread and infection.