Human nuclear protein positive cofactor PC4 is a DNA-binding protein, and plays an important role in the early response to DNA damage by recognizing single-stranded oligodeoxynucleotide domain and facilitating the subsequent steps of DNA repair. Our group previously discovered that PC4 selectively binds to a double-stranded oligodeoxynucleotide (dsODN) damaged by a trans-platinum anticancer complex, trans-[PtCl2(NH3)(thiazole)] (trans-PtTz). However, the molecular basis of this unique recognition and interaction remained unclear. In this work, amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to dissect the interaction interface between PC4 and a 15-mer double-stranded oligodeoxynucleotide crosslinked by trans-PtTz (trans-PtTz-dsODN). Global deuterium uptake suggested a 1:1 binding stoichiometry of PC4 to trans-PtTz-dsODN. Local deuterium uptake revealed that the flexible N-terminal loop and the β3 – β5-sheet played key roles in the recognition and interaction between PC4 and trans-PtTz-dsODN. In order to locate the key amino acid residues, molecular dynamics simulation was employed, demonstrating that PC4 binds to the trans-PtTz-dsODN at the minor groove via strong H-bonds with the nucleobases of the complementary strand. The minor groove width was broadened to adapt for the binding of PC4. Arg86 residue in PC4 was shown to dominate the recognition and interaction, which was verified by electrophoretic mobility shift assays. This work profiles the detailed interaction mechanism of PC4 with trans-PtTz damaged dsODN, and the combination use of HDX-MS and molecular dynamics simulation provides a new paradigm for the future research of the cellular response to the platinum induced DNA damage.