Using monolayers of intestinal (Caco-2) cells, we reported that upregulation of iNOS and overproduction of reactive nitrogen metabolites are key to oxidant-induced loss of barrier integrity and that EGF protects against this injury by preventing the oxidation of the microtubule cytoskeleton. Expression of the PKC-zeta () isoform was required for protection. For the current investigation, we hypothesized that PKC- activation and iNOS down-regulation are key in EGF protection against oxidative injury. Methods: Wild type (WT) Caco-2 cells were incubated with H₂O₂ (0.5 mM) ± EGF or PKC modulators. Other WT Caco-2 cells were transfected to stably over-express PKC- or to inhibit its expression and then pretreated with EGF or a PKC activator (OAG) prior to oxidant. Results: {A} Relative to WT monolayers exposed to oxidant alone, pretreatment with EGF protected monolayers by 1) increasing PKC- activity; 2) decreasing six iNOS related variables [iNOS activity & protein levels, NO levels, oxidative stress, tubulin oxidation & nitration]; 3) increasing stable polymerized tubulin; 4) maintaining the cytoarchitectural integrity of microtubules; and 5) enhancing barrier integrity. {B} Relative to WT cells exposed to oxidant, monolayers of transfected cells stably over-expressing PKC- (+2.9 fold) were protected as indicated by decreases in all measures of iNOS driven pathways as well as enhanced stability of the microtubule cytoarchitecture and of barrier function. Over-expression induced inhibition of iNOS was OAG-independent, although EGF potentiated this protection. {C} Anti-sense inhibition of PKC- expression (-95%) prevented all measures of EGF protection against iNOS upregulation. Conclusions: (1) EGF protects against oxidative disruption of the intestinal barrier integrity by stabilizing the cytoskeleton, in large part, through the activation of PKC- and down-regulation of iNOS; (2) Activation of PKC- is by itself required for cellular protection against oxidative stress of iNOS up-regulation. (3) We have thus discovered novel biologic functions - suppression of the iNOS driven reactions and of cytoskeletal oxidation- among the atypical PKC isoforms. Accordingly, increasing PKC- signaling is a potentially novel therapeutic strategy in intestinal disorders such as inflammatory bowel disease (IBD) that are associated with oxidative stress injury.