Using monolayers of intestinal (Caco-2) cells, we previously found that upregulation of iNOS is key to oxidant-induced disruption of barrier integrity and that EGF protects against this disruption by stabilizing the cytoskeleton. Expression of phospholipase C- (PLC-) appears to be essential for monolayer maintenance. In the current investigation, we hypothesized that PLC- activation is essential in EGF-mediated protection against iNOS up-regulation and the consequent cytoskeletal oxidation and disarray and monolayer barrier disruption. Intestinal cells were transfected with varying levels (1-5 µg) of DNA to either stably over-express PLC- or to inhibit its activation and then pretreated with EGF ± oxidant (H₂O₂). Wild type (WT) intestinal cells were treated similarly. Results: {A} Relative to WT monolayers exposed to oxidant, pretreatment with EGF protected monolayers by: 1) increasing native PLC- activity; 2) decreasing 6 iNOS related variables [iNOS activity and protein, NO levels, oxidative stress, actin oxidation and nitration]; 3) increasing stable F-actin; 4) maintaining architecture of actin cytoskeleton; 5) enhancing barrier integrity. {B} Relative to WT cells exposed to oxidant, monolayers of transfected cells over-expressing PLC- (+2.3 fold) were protected as indicated by decreases in all measures of iNOS driven pathway and enhanced actin and barrier integrity. Over-expression induced inhibition of iNOS was potentiated by low doses of EGF. {C} Stable inhibition of PLC- substantially prevented all measures of EGF protection against iNOS upregulation. Conclusions: 1) EGF protects against oxidative stress disruption of the intestinal barrier integrity by stabilizing the F-Actin, in large part, through the activation of PLC- and down-regulation of iNOS pathway; 2) Activation of PLC- is by itself essential for cellular protection against oxidative stress of iNOS up-regulation; 3) The ability to suppress iNOS driven reactions and cytoskeletal oxidation and disassembly is a novel mechanism not previously attributed to the PLC family of isoforms.