Enterocytes maintain fluid-electrolyte homeostasis by keeping a tight barrier and regulating ion channels. Carbon monoxide (CO), a product of heme degradation, modulates electrolyte transport in kidney and lung epithelium, but its role in regulating intestinal fluid-electrolyte homeostasis has not been studied. The major source of endogenous CO formation comes from the degradation of heme via heme oxygenase. We hypothesized that heme activates electrolyte transport in intestinal epithelial cells. Basolateral hemin treatment increased baseline Caco-2 cell short circuit currents (I_sc) two-fold (control=1.96±0.14 µA/cm²vs hemin=4.07±0.16 µA/cm², p<0.01), apical hemin had no effect. Hemin-induced I_sc was caused by Cl^- secretion because it was inhibited in Cl^- free medium, with ouabain, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or 4,4'-diisothiocyanostilbene-2',2-disulphonic acid (DIDS). Apical electrogenic Na⁺ channel (ENaC) inhibitor, benzamil had no effect on hemin-induced I_sc. Hemin did not alter the ability of Caco-2 cells to respond maximally to forskolin, but a soluble guanylate cyclase inhibitor, [1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of hemin. A CO releasing molecule, tricarbonyldichlororuthenium II induced active Cl^- secretion that was also inhibited with ODQ. We conclude that hemin induces active Cl^- secretion in Caco-2 cells via a cyclic GMP-dependent pathway. These effects are probably the consequence of CO formation. Heme and CO may be important regulators of intestinal fluid-electrolyte homeostasis.