Reactive oxygen species (ROS) are key mediators in a number of inflammatory conditions, including inflammatory bowel disease (IBD). ROS, including hydrogen peroxide (H₂O₂), modulate intestinal epithelial ion transport and are believed to contribute to IBD-associated diarrhea. Intestinal crypt fluid secretion, driven by electrogenic Cl^- secretion, hydrates and sterilizes the crypt, thus reducing bacterial adherence. Here, we show that pathophysiological concentrations of H₂O₂ inhibit Ca²⁺-dependent Cl^- secretion across T₈₄ colonic epithelial cells by elevating cytosolic Ca²⁺, which contributes to activation of two distinct signaling pathways. One involves recruitment of the Ca²⁺-responsive kinases, Src and Pyk-2, as well as extracellular signal-regulated kinase (ERK). A separate pathway recruits p38 MAP kinase and phosphoinositide 3-kinase (PI3-K) signaling. The ion transport response to Ca²⁺-dependent stimuli is mediated in part by K⁺ efflux through basolateral K⁺ channels and Cl^- uptake by the Na⁺-K⁺-2Cl^- cotransporter, NKCC1. We demonstrate that H₂O₂ inhibits Ca²⁺-dependent basolateral K⁺ efflux and also inhibits NKCC1 activity independently of inhibitory effects on apical Cl^- conductance. Thus, we have demonstrated that H₂O₂ inhibits Ca²⁺-dependent Cl^- secretion through multiple negative regulatory signaling pathways and inhibition of specific ion transporters. These findings increase our understanding of mechanisms by which inflammation disturbs intestinal epithelial function and contributes to intestinal pathophysiology.—Chappell, A. E., Bunz, M., Smoll, E., Dong, H., Lytle, C., Barrett, K. E., McCole, D. F. Hydrogen peroxide inhibits Ca²⁺-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins.