The present study analyzed the transepithelial transport of the dietary anti-inflammatory peptide, γ-glutamyl valine (γ-EV). γ-EV is naturally found in dry edible beans. Our previous study demonstrated the anti-inflammatory potency of γ-EV against vascular inflammation at a concentration of 1mM, and that it can transport with the apparent permeability coefficient (P) of 1.56 × 10 ± 0.7 × 10 cm/s across the intestinal Caco-2 cells. The purpose of the current study was to explore whether the permeability of the peptide could be enhanced and to elucidate the mechanism of transport of γ-EV across Caco-2 cells. The initial results indicated that γ-EV was nontoxic to the Caco-2 cells up to 5 mM concentration and could be transported across the intestinal cells intact. During apical-to-basolateral transport, a higher peptide dose (5 mM) significantly ( < 0.01) enhanced the transport rate to 2.5 × 10 ± 0.6 × 10 cm/s. Cytochalasin-D disintegrated the tight-junction proteins of the Caco-2 monolayer and increased the P of γ-EV to 4.36 × 10 ± 0.16 × 10 cm/s ( < 0.001), while theaflavin 3'-gallate and Gly-Sar significantly decreased the P ( < 0.05), with wortmannin having no effects on the peptide transport, indicating that the transport route of γ-EV could be via both PepT1-mediated and paracellular.