Many studies have implicated F-actin in the regulation of gastric acid secretion using cytochalasin D (CD) to disrupt apical actin filaments in oxyntic cells. However, it is known that CD also affects mucosal permeability by disrupting tight junction structure. Here we investigate the contribution of F-actin to mucosal permeability and acid secretion in the stomach using CD. Stomachs were mounted in Ussing chambers and acid secretion (stimulated or inhibited), transepithelial resistance (TER), mannitol flux, bicarbonate transport, and dual mannitol/sodium fluxes were determined with or without CD. H⁺ back-diffusion was predicted from its diffusion coefficient. Incubation with CD resulted in a significant reduction in stimulated acid secretion. TER was unchanged in stimulated but significantly reduced in inhibited tissues. Mannitol flux, bicarbonate transport, and H⁺-back-diffusion increased significantly with CD. However, the rates of bicarbonate and H⁺-flux were not large enough to account for the inhibition of acid secretion. These findings demonstrate that actin filaments regulate paracellular permeability and play an essential role in the regulation of acid secretion in the stomach.