<b>Objectives</b><br/> We aimed at evaluating the virulence of atypical <i>Shigella flexneri</i> II:(3)4,7(8) by DNA microarray and invasion assay.<br/><b>Methods</b><br/> We used a customized <i>S. flexneri</i> DNA microarray to analyze an atypical <i>S. flexneri</i> II:(3)4,7(8) gene expression profile and compared it with that of the <i>S. flexneri</i> 2b strain.<br/><b>Results</b><br/> Approximately one-quarter of the atypical <i>S. flexneri</i> II:(3)4,7(8) strain genes showed significantly altered expression profiles; 344 genes were more than two-fold upregulated, and 442 genes were more than 0.5-fold downregulated. The upregulated genes were divided into the category of 21 clusters of orthologous groups (COGs), and the “not in COGs” category included 170 genes. This category had virulence plasmid genes, including the <i>ipa-mxi-spa</i> genes required for invasion of colorectal epithelium (type III secretion system). Quantitative reverse-transcription polymerase chain reaction results also showed the same pattern in two more atypical <i>S. flexneri</i> II:(3)4,7(8) strains. Atypical <i>S. flexneri</i> II:(3)4,7(8) showed four times increased invasion activity in Caco-2 cells than that of typical strains.<br/><b>Conclusion</b><br/> Our results provide the intracellularly regulated genes that may be important for adaptation and growth strategies of this atypical <i>S. flexneri</i>.