COL17A1 is predominantly expressed in skin epithelial cells and primarily localized within hemidesmosomes. It plays an essential role in epidermal-dermal attachment. Consequently, a recombinant human-like COL17A1 protein (rhCOL17) with low molecular weight and high biocompatibility presents a promising and competitive biomaterial. The aim of this study is to gain more insight into the biological functions and underlying molecular mechanisms of rhCOL17, which primarily consists of amino acid residues Gly659-Leu720. Using a combination of surface plasmon resonance (SPR) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified the interacting partner proteins of rhCOL17 in HaCaT cells. These included several collagens, integrins, and cell polarity proteins. Upon rhCOL17 treatment, the expression levels of laminin-332, integrin β1, and the cell polarity proteins PAR-3 and PAR-6B were upregulated, while the PRKCZ, AKT, and TGF-β1 signaling pathways were activated. Furthermore, rhCOL17 was found to promote cell proliferation and mitigate UV radiation-induced damage, partly by modulating these interacting proteins and their associated signaling pathways. Additional analyses using AlphaFold2 and molecular dynamics simulations revealed that the rhCOL17 peptide bound stably and tightly to the canonical ligand-binding site between the integrin α3 and β1 subunits. These findings highlight the potential versatility and applications of rhCOL17 in the field of antiaging.