Coxsackie B4 (CV-B4), is a major cause of viral myocarditis, dilated cardiomyopathy, and pancreatitis. Like other human enteroviruses, CV-B4 is ubiquitous, excreted in the stool, transmitted by fecal-oral route, and persists in the environment. In the context of studies on CV-B4 infection, it is important to investigate how this virus can be eliminated and to show the possibility of contamination risk with a CV-B4 E2 infected Swiss albino mice. Swiss albino female mice were inoculated with CV-B4 E2 strain and divided in two groups: the first was intraperitoneally (I.P.) infected; the second was orally infected. In order to study the CV-B4 E2 infection in mice, total RNA was extracted from thymus, spleen, pancreas, and intestine, and viral genome was detected using semi-nested (RT-PCR). To further demonstrate infection or immunization of mice, Sera obtained from infected mice were assayed in vitro for their neutralizing antibody. To detect virus in stool of infected mice, stool samples were collected at different post-infection (p.i.) times. Neutralizing antibodies were detectable all along the follow-up period (Day 0, 1, 3, 7, 9, 17, 22, 30, 45, 60 p.i.) in I.P and oral infected mice. Our results showed that when mice were inoculated successively at day 0 and day 8, neutralizing activity was increased in I.P route more than in the oral route. Viral isolation in HEp-2 cells showed negative results. Stool viral analyses reveal a low detection of the CV-B4 E2 genome for all infected mice. In conclusion, our experiments demonstrated that there are no risks linked with the stool of CV-B4 E2 of Swiss albino mice. It would be interesting to characterize the inhibitors of the virus infectivity in these biological samples (stool) and investigate their targets and mechanisms of action.