Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in lowto- middle-income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF that are prime targets as vaccine antigens. CFA/I is a major CF that is associated with ETEC isolates that cause moderate-to-severe diarrhea and has been shown to play an important role in ETEC pathogenesis in in vitro and in vivo studies. The Global Enteric Multicenter Study finding that 78% of CFA/I-expressing ETEC also encode the minor CF CS21 prompted investigation of the potential combined role of these two CFs. Western blots and electron microscopy demonstrated growth media-dependent and clinical isolatedependent differences in CFA/I and CS21 expression. The critical role of CFA/I, but not CS21, in adherence was demonstrated using the human enteroid model and a series of CFA/I- and CS21-specific mutants, suggesting an alternative role for CS21 in ETEC pathogenesis. Furthermore, only anti-CFA/I antibodies inhibited adherence by global ETEC isolates expressing CFA/I and CS21. Delivery of ST and resulting cGMP secretion was measured in apical and basolateral supernatants from infected enteroid monolayers, and strain-specific ST delivery and time-dependent cGMP production was observed. Interestingly, cGMP levels were similar across wildtype and CF-deficient strains, reflecting the limitations of this static infection model that lacks flow and stretch and is exclusively aerobic. Despite adherence by ETEC and delivery of ST, the enteroid monolayer integrity was not disrupted, as shown by the increase in transepithelial resistance and the lack of inflammatory cytokines produced during infection. Altogether, these data demonstrate that targeting CFA/I in global clinical CFA/I-CS21 strains is sufficient for adherence inhibition, supporting a vaccine strategy that focuses on blocking major CFs. In addition, the enteroid model has significant utility for the study of ETEC pathogenesis.