Dysregulated epithelial secretory function can lead to the clinical manifestation of diarrhoea. Intestinal fluid secretion is driven by active Cl- ion secretion and is dependent on the availability of O2 for generation of cellular energy. Hydroxylases are the primary intracellular sensors of O2 availability, and we have previously shown that hydroxylase inhibition attenuates colonic epithelial secretory capacity. Here, we sought to investigate the molecular mechanisms involved and to test the efficacy of hydroxylase inhibitors in preventing diarrhoea in vivo. Dimethyloxallyl glycine (DMOG) was used to inhibit hydroxylase activity. Cl- secretion was measured as changes in short circuit current across monolayers of T84 cells or muscle-stripped segments of mouse colon. Western blotting and biotinylation techniques were used to assess protein expression. DMOG effects were also tested in vivo in a mouse allergic (ovalbumin) model of diarrhoea. As previously reported, treatment of T84 cells with DMOG (1 mM) reduced Cl- secretory responses to the Ca2+ and cAMP-dependent agonists, carbachol (CCh) and forskolin (FSK), to 20.2 ± 2.6% and 38.8 ± 4.8% of controls, respectively (n=16; p ≤ 0.001). To determine molecular mechanisms involved we analysed the activity of transport proteins comprising the Cl- secretory pathway. Hydroxylase inhibition did not alter apical Cl- or basolateral K+ conductances but significantly reduced the activity of the Na+/K+ATPase, the energy-dependent step of Cl- secretion, to 42.7% ± 5.5% of controls (n=5; p ≤ 0.01). However, DMOG did not alter total cellular or surface expression of the ATPase. Cellular ATP, required to drive pump activity, was not affected by DMOG. Intraperitoneal injection of mice with DMOG (320 mg/kg; 24 hrs) attenuated colonic secretory responses to CCh and FSK to 54.8 ± 6.1% (n=11; p ≤ 0.001) and 72.4 ± 11.5% (n=11; p ≤ 0.05) of those of controls, respectively. Furthermore, in an in vivo mouse model of allergic diarrhoea, DMOG pre-treatment reduced the occurrence of diarrhoea by 80% compared to controls (n=10). Our studies show that hydroxylase inhibition in colonic epithelial cells inhibits secretory function both in vitro and in vivo. This antisecretory effect is mediated by attenuation of Na+/K+-ATPase pump activity without alterations in expression of the protein. Our data suggest that hydroxylases present a promising target for the development of novel anti-diarrhoeal agents that act by directly modulating intestinal epithelial transport protein function.