In many gut chronic inflammatory conditions, intestinal epithelium (IE) is deprived of the protection of the mucus secreted by IE-specialized cells. In these events, bleeding and subsequent lysis of erythrocytes are common. This may lead to the release of high amounts of heme in the intestinal lumen, which interacts with IE. Previous works from our group have shown that heme itself is a proinflammatory molecule, activating a number of phlogistic signaling events in a nicotinamide adenine dinucleotide phosphate oxidase (NADPHox)-dependent manner. In this study, we aim to evaluate the effects of heme upon a well-established nontransformed small intestine epithelial cell lineage (IEC 6). Our results show that free heme evokes intracellular reactive oxygen species (ROS) production by IEC 6 cells, which is inhibited both by pharmacological inhibition with diphenyleneiodonium (10 μM), a NADPHox inhibitor, and small interfering RNA-mediated suppression of NOX1, a constitutive NADPHox isoform present in intestinal epithelial cells. Focal adhesion kinase phosphorylation and actin cytoskeleton polymerization are also induced by heme in a NADPHox-dependent manner. Heme increases monolayer permeability and redistributes key modulators of cell-cell adhesion as zona occludens-1 and E-cadherin proteins via NADPHox signaling. Heme promotes IEC 6 cell migration and proliferation, phenomena also regulated by NADPHox-derived ROS. Heme, in NADPHox-activating concentrations, is able to induce mRNA expression of IL-6, a cytokine implicated in inflammatory and tumorigenic responses. These data indicate a prominent role for heme-derived signaling in the pathophysiology of intestinal mucosa dysfunction and address an important role of NADPHox activity on the pathogenesis of intestinal inflammatory conditions.