<p>We aimed to develop an experimental model system using human intestinal organoids to examine the invasion abilities of different <i>Shigella</i> species ((<i>S. dysenteriae</i>, <i>S. flexneri</i>, and <i>S. sonnei</i>). Human intestinal organoids (enteroids) were developed and established using human intestinal biopsies. Three distinct species of <i>Shigella</i> were used to infect enteroids<i>.</i> The ability to invade was checked by gentamicin protection assay. We visualized the entry of <i>Shigella</i> and morphological changes induced by bright field microscopy and immunofluorescence microscopy. We studied the differential expression of the <i>ipa</i>H gene in infected human intestinal organoids and correlated it with the degree of invasiveness. Our results demonstrated that different species of <i>Shigella</i> vary in their invasive ability, with <i>S.dysenteriae</i> being more invasive than <i>S.flexneri</i> and <i>S.sonnei</i>. Our data showed that the intestinal organoid model is feasible for studying <i>Shigella</i> pathogenesis. This model would be suitable for discovering new antigens and testing vaccines against <i>Shigella</i>. This investigation holds significant promise for improving our understanding of <i>Shigella</i> pathogenesis. Furthermore, human intestinal organoids provide a rapidly replicable model system model system of <i>Shigella</i> pathogenesis due to their ability to be expanded, genetically modified and cryogenically maintained. Insights gained from this study could have broader implications for addressing diarrheal illnesses, particularly in regions where <i>Shigella</i> infections pose a significant public health burden. To our knowledge, this is the first study from India to utilize human-derived intestinal organoids to systematically evaluate the differential invasion capacity, epithelial damage, and virulence gene expression induced by clinically prevalent <i>Shigella</i> serotypes.</p> Graphical Abstract <p></p>