Enterotoxigenic Escherichia coli (ETEC) strains are characterized by colonization surface antigens (CSs) that play a pivotal role in their virulence by facilitating gut colonization, the initial step in diarrheal pathogenesis. Among these CSs, type IV pili, also known as longus, are implicated in bacterial self-aggregation, biofilm formation, and resistance to environmental stresses. CS21, a specific CS expressed by ETEC, mediates adherence to the primary epithelium during infection. In this study, we investigated the role of CS21 in ETEC pathogenesis using in vitro and in vivo models. ETEC isolates expressing CS21, including both wild-type and mutant strains, were evaluated for their adherence to pig intestinal cell lines. Additionally, neonatal mice were employed as infection models to assess the contribution of CS21 to ETEC pathogenicity. Adherence assays revealed CS21-dependent attachment of ETEC to intestinal cells, with purified LngA, a component of CS21, effectively competing with CS21-expressing ETEC for cellular attachment, an effect reversed by anti-LngA antibodies. Furthermore, in vivo studies demonstrated a significant reduction in the pathogenicity of CS21-deficient ETEC strains upon intravenous injection into newborn DBA/2 mice. These findings underscore the critical role of CS21 in mediating ETEC infections, highlighting its potential as a target for therapeutic interventions