Shigellosis is a significant type of diarrhea that causes 160,000 deaths annually in a global scale. The mortality occurs mainly in children less than 5 years of age. No licensed vaccine is available, and conventional efforts for developing an effective and safe vaccine against shigellosis have not been succeeded yet. The reverse vaccinology is a novel promising method that screens genome or proteome of an organism for finding new vaccine candidates. In this study, through reverse vaccinology approach, new vaccine candidates against Shigella flexneri were identified and experimentally evaluated. Proteomes of S. flexneri were obtained from UniProt, and then outer membrane and extracellular proteins were predicted and selected for the evaluation of transmembrane domains, protein conservation, host homology, antigenicity, and solubility. From 103 proteins, 7 high-scored proteins were introduced as novel vaccine candidates, and after Band T-cell epitope prediction, the best protein was selected for experimental studies. Recombinant protein was expressed, purified, and injected to BALB/c mice. The adhesion inhibitory effect of sera was also studied. The immunized mice demonstrated full protection against the lethal dose challenge. The sera remarkably inhibited S. flexneri adhesion to Caco-2 epithelial cells. The results indicate that identified antigen can serve for vaccine development against shigellosis and support reverse vaccinology for discovering novel effective antigens. Key points • Seven Shigella new antigens were identified by reverse vaccinology (RV) approach. • The best antigen experimented demonstrated full protection against lethal dose. • In vivo results verified RV analyses and suggest FimG as a new potent vaccine candidate.