Purpose <p>To improve bioactive peptide absorption and bioavailability, in-depth understanding of their transport across the intestinal epithelium is crucial. In this study, we used Caco-2 cell monolayers to investigate the transepithelial transport of Trp-Tyr-Thr (WYT), a novel hemp protein-derived Angiotensin-I converting enzyme (ACE) peptide.</p> Methods <p>Firstly, Caco-2 cell monolayer model was established and then various cell modulators (Gly-Pro, sodium azide, wortmannin and cytochalasin D) were used to study WYT transport.</p> Results <p>Our results demonstrated that WYT maintained its stability for more than 2 days and 2&#xa0;h in Hank’s balanced salt solution and on the apical surface of Caco-2 cells, respectively. WYT was transported intact across Caco-2 cell monolayers with an apparent permeability coefficient (P_app) of 1.03 × 10⁻⁷ cm/s. Our transport route evaluation revealed that Gly-Pro, a peptide transporter 1 (PepT1) substrate, significantly affected transepithelial permeability, suggesting that it mediated WYT transport across Caco-2 cell monolayers. Moreover, the ATPase inhibitor sodium azide significantly reduced the P_app of WYT, while the tight junction disruptor cytochalasin D and the transcytosis inhibitor wortmannin did not significantly affect WYT permeability.</p> Conclusion <p>In summary, the results of our study indicate that WYT transport across Caco-2 cell monolayers occurs energy-dependently via the PepT1 pathway. It contributes to the study of hemp protein-derived bioactive peptide in lowering blood pressure and supports the development of bioactive peptide products.</p>