Cryptosporidium sp. parasitizes intestinal epithelium, resulting in enterocyte loss, villous atrophy, and malabsorptive diarrhea. We have shown that mucosal expression of iNOS is increased in infected piglets and inhibition of iNOS in vitro has no short-term effect on barrier function. Nitric oxide exerts inhibitory effects on a variety of pathogens; nevertheless, the specific sites of iNOS expression, pathways of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear. Here we examined the location, mechanism of induction, specificity, and consequence of iNOS expression using an in vivo model of C. parvum infection in neonatal piglets. In acute C. parvum infection, iNOS expression predominated in the villous epithelium, was NF-B-dependent, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion but was without detriment to maintenance of mucosal barrier function. Intensified parasitism could not be attributed to attenuated fluid loss or changes in epithelial proliferation or replacement rate as iNOS inhibition did not alter severity of diarrhea, piglet hydration, Cl^- secretion or kinetics of BrdU-labeled enterocytes. These findings suggest that induction of iNOS represents a non-specific response of the epithelium that mediates enterocyte defense against C. parvum infection. Inducible NOS did not contribute to the pathogenic sequelae of C. parvum infection.