Extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple interactions with various gut epithelial components. For instance, GAPDH inLactobacillus johnsoniiMG cells interacts with junctional adhesion molecule-2 (JAM-2) in Caco-2 cells and enhances tight junctions. However, the specificity of GAPDH toward JAM-2 and its role in the tight junctions in Caco-2 cells remain unclear. In the present study, we assessed the effect of GAPDH on tight junction regeneration and explored the GAPDH peptide fragments required for interaction with JAM-2. GAPDH was specifically bound to JAM-2 and rescued H2O2-damaged tight junctions in Caco-2 cells, with various genes being upregulated in the tight junctions. To understand the specific amino acid sequence of GAPDH that interacts with JAM-2, peptides interacting with JAM-2 andL. johnsoniiMG cells were purified using HPLC and predicted using TOF–MS analysis. Two peptides, namely11GRIGRLAF18at the N-terminus and323SFTCQMVRTLLKFATL338at the C-terminus, displayed good interactions and docking with JAM-2. In contrast, the long peptide52DSTHGTFNHEVSATDDSIVVDGKKYRVYAEPQAQNIPW89was predicted to bind to the bacterial cell surface. Overall, we revealed a novel role of GAPDH purified fromL. johnsoniiMG in promoting the regeneration of damaged tight junctions and identified the specific sequences of GAPDH involved in JAM-2 binding and MG cell interaction.