Colonization surface antigens (CSs) represent key virulence-associated factors of enterotoxigenic Escherichia coli (ETEC) strains. They are required for gut colonization, the first step of the diarrhoeal disease process induced by these bacteria. One of the most prevalent CSs is CS21, or longus, a type IV pili associated with bacterial self-aggregation, protection against environmental stresses, biofilm formation and adherence to epithelial cell lines. The objectives of this study were to assess the role of CS21 in adherence to primary intestinal epithelial cells and to determine if CS21 contributes to the pathogenesis of ETEC infection in vivo. We evaluated adherence of a CS21-expressing wild-type ETEC strain and an isogenic CS21-mutant strain to pig-derived intestinal cell lines. To determine the role of CS21 in pathogenesis we used the above ETEC strains in a neonatal mice challenge infection model to assess mortality. Quantitative adherence assays confirmed that ETEC adheres to primary intestinal epithelial cells lines in a CS21-dependent manner. In addition, the CS21-mediated ETEC adherence to cells was specific as purified LngA protein, the CS21 major subunit, competed for binding with the CS21-expressing ETEC while specific anti-LngA antibodies blocked adhesion to intestinal cells. Neonatal DBA/2 mice died after intra-stomach administration of CS21-expressing strains while lack of CS21 expression drastically reduced the virulence of the wild-type ETEC strain in this animal model. Collectively these results further support the role of CS21 during ETEC infection and add new evidence on its in vivo relevance in pathogenesis.