enterotoxin (CPE) contributes to diarrhea and an often-lethal enterotoxemia. CPE action starts when it binds to claudin receptors, forming a small complex (90 kDa). Six small complexes then oligomerize to create prepores, followed by insertion of beta-hairpins from CPE to form beta-barrel pores named CH-1 or CH-2. Of the ~27 members of the human claudin protein family, only some bind CPE. However, both receptor claudins and the nonreceptor claudin-1 (CLDN-1) are associated with the small and CH-1/CH-2 CPE complexes. Therefore, this study evaluated whether claudin-1 affects CPE action by generating a CLDN-1 null mutant in Caco-2 cells using CRISPR-Cas9. Compared to wild-type Caco-2 cells, paracellular permeability of the CLDN-1 mutant was significantly enhanced, suggesting that claudin-1 may reduce CPE absorption during enterotoxemia. The CLDN-1 mutant was also markedly more sensitive than wild-type Caco-2 cells to apically-applied CPE. The mechanism behind this increased sensitivity involved higher CPE binding by the CLDN-1 mutant vs. wild-type Caco-2 cells, which led to more CH-1/CH-2 complex formation. However, the CH-1/CH-2 complexes formed by the CLDN-1 mutant were less stable or trypsin resistant than those of wild-type cells. These results indicate that, although a nonreceptor, CLDN-1 positively and negatively influences CPE action.