Shigella is one of the main pathogens causing diarrheal disease, and is associated with high morbidity and mortality in developing countries. Previous clinical data and animal studies have shown that the outcomes of oral and peritoneal infections of Shigella differ, and that the latter is more serious. Furthermore, a variety of pathogenic bacteria are known to cause changes in intestinal flora after infection, and the influence of Shigella infection on intestinal flora remains poorly understood. In the present study, the 16S rRNA high-throughput sequencing method was used to compare the changes in gut microbiota profiles in feces of mice infected with Shigella via two routes. In addition, the present study investigated the association between the differences in infection performance and bacterial communities. The present results suggested that the intraperitoneal route induced a distinct decrease in α-diversity in the fecal microbiota when compared to the control at a later time, while the effect of the oral route on α-diversity was not obvious. Oral infection of Shigella had a rapid and significant effect on gut microbiota, mainly causing a decreased abundance of Lactobacillus and an increased abundance of Prevotella and Escherichia/Shigella in the early stage of infection. By contrast, the effect of intraperitoneal infection on the gut microbiota was relatively slow and small. The principal coordinate analysis results suggested that the dynamic profile of gut microbiota between the two infective routes was consistent with the infection process. Probiotics, such as Lactobacillus reuteri and Faecalitalea exhibited significantly reduced abundance after Shigella infection. Collectively, the present results suggested that gut microbiota may play a pivotal role in the pathogenesis of Shigella infection. Future studies should investigate the effect of Shigella infection on the interaction between pathogenic bacteria and intestinal flora. The present results suggested that the use of probiotics may facilitate the prevention and treatment of shigellosis.