Folic acid (FA) is a vitamin essential for normal cellular functions, growth, and development. Because humans cannot synthesize this micronutrient, it must be obtained from dietary sources through intestinal absorption. The intestinal tract is a major target for oxidative stress. Our aim was to investigate the effect of oxidative stress upon the uptake of FA by Caco-2 cells. Oxidative stress was induced by exposure of the cells to tert-butyl hydroperoxide (TBH) for 1 h. TBH (3,000 μM) induced an increase in biomarkers of oxidative stress, while maintaining cell viability and proliferation. In relation to the apical uptake of (3)H-FA, TBH (3,000 μM) reduced the cellular accumulation of (3)H-FA (10 nM), although the characteristics (kinetics, pH dependence, and inhibitory profile) of (3)H-FA uptake were not changed. This effect was associated with a decrease in the mRNA steady-state levels of proton-coupled folate transporter and folate receptor alpha and of the efflux transporter multidrug resistance protein 2. Moreover, TBH (3,000 μM) did not affect the noncarrier-mediated apical uptake of (3)H-FA. Finally, the effect of TBH upon (3)H-FA apical uptake was not dependent on protein kinase A, protein kinase C, mitogen-activated protein kinases, phosphoinositide 3-kinase, nuclear factor kappa B, and protein tyrosine kinases, but was completely prevented by dietary polyphenols (resveratrol, quercetin, and EGCG). These results suggest that oxidative stress at the intestinal level may result in a reduction in the intestinal absorption of dietary FA and that polyphenolic dietary components may offer protection against oxidative stress-induced inhibition of intestinal FA absorption.