We investigated the effects of DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) on the Cl^- secretory response of the mouse jejunum using the Ussing short-circuit current (I_sc) technique. DCEBIO stimulated a concentration-dependent, sustained increase in I_sc (EC₅₀ 41 ± 1 µM). Pretreating tissues with 0.25 µM forskolin reduced the concentration-dependent increase in I_sc by DCEBIO and increased the EC₅₀ (53 ± 5 µM). Bumetanide blocked (82 ± 5%) the DCEBIO-stimulated I_sc consistent with Cl^- secretion. DCEBIO was a more potent stimulator of Cl^- secretion than its parent molecule 1-ethyl-2-benzimidazolinone. Glibenclamide or NPPB reduced the DCEBIO-stimulated I_sc by >80% indicating the participation of CFTR in the DCEBIO-stimulated I_sc response. Clotrimazole reduced DCEBIO-stimulated I_sc by 67 ± 15% suggesting the participation of the intermediate conductance Ca²⁺-activated K⁺ channel (IK_Ca) in the DCEBIO-activated I_sc response. In the presence of maximum forskolin (10 µM), the DCEBIO response was reduced and biphasic reaching a peak response of I_sc of 43 ± 5 µA/cm² and then falling to a steady-state response of 17 ± 10 µA/cm² compared with DCEBIO control tissues (61 ± 6 µA/cm²). The forskolin-stimulated I_sc in the presence of DCEBIO was reduced compared to forskolin control tissues. Similar results were observed with DCEBIO and 8-Br-cAMP where adenylate cyclase was bypassed. H89, a PKA inhibitor, reduced the DCEBIO-activated I_sc providing evidence that DCEBIO increased Cl^- secretion via a cAMP/PKA-dependent manner. These data suggest that DCEBIO stimulates Cl^- secretion of the mouse jejunum and that DCEBIO targets components of the Cl^- secretory mechanism.