Marine-derived collagen peptides exhibit potent intestinal barrier protection; however, their gastrointestinal fate and molecular targets remain unclear, limiting their practical applications. This study investigated the digestive stability and transepithelial transport of GPSGPQGSR, a mucoprotective peptide from Alaska pollock (Gadus chalcogrammus) skin, using simulated gastrointestinal digestion, a Caco-2 cell transport model, and an UPLC-QTOF-MS/MS. The results showed that GPSGPQGSR was a digestion-resistant peptide that reached the intestinal epithelium intact. Although brush border membrane enzymes partially hydrolysed the peptide, 42.16% of intact GPSGPQGSR remained in the luminal compartment after 2 h of incubation. No intact peptide was detected in the basolateral compartment. Molecular docking and 100 ns molecular dynamics simulations identified TLR2 (−14.936 kcal/mol) and PAR2 (−10.154 kcal/mol) as high-affinity extracellular targets of GPSGPQGSR, with stable peptide–receptor interactions and extensive hydrogen bonding networks between the peptide and each receptor (RMSD of 1.8 Å and 2.2 Å, respectively). Pharmacological blockade of TLR2 or PAR2 abolished the protective effects of GPSGPQGSR. These findings demonstrate that GPSGPQGSR acts as a digestion-resistant extracellular signalling peptide that reaches the intestinal epithelium intact and protects barrier function through apical TLR2 and PAR2, providing a mechanistic basis for the rational development of marine collagen peptides for improving intestinal health.