ABSTRACTMajor histocompatibility complex class I‐bound antigenic peptides generated in the cytosol are translocated into the ER by TAP. In the present study, the physical association of HSC73 with TAP in human lymphoblastoid T1 cells was demonstrated. The dissociation was induced in the presence of 10 mM ATP, indicating that the ADP‐binding form of HSC73 might be associated with TAP. We found that HSC73‐binding immunosuppressant, MeDSG disrupted the HSC73‐TAP association, whereas it did not affect the binding of HSC73 to a substrate protein. MHC class I expression on the cell surface was also downregulated. Then, the effect of MeDSG on the TAP‐mediated ER translocation was examined using two homologous model peptides, NGT‐Bw4 and NGT‐Bw6, which had distinct binding affinity to HSC73. Although high‐affinity peptide NGT‐Bw4 was translocated by TAP, low‐affinity peptide NGT‐Bw6 was not. The TAP‐dependent translocation of NGT‐Bw4 was abolished in the presence of MeDSG. Decreased presentation on the cell surface was shown for the human leukocyte antigen (HLA)‐A31‐restricted natural antigenic peptide F4.2, which had high affinity to HSC73, in the presence of MeDSG. It was indicated that disruption of the HSC73‐TAP association resulted in inhibition of TAP‐dependent translocation of HSC73‐bound peptides. Our findings highlighted an important role of HSC73 for feeding antigenic peptides to TAP, and suggested a possibility that a synthetic polyamine might inhibit the function of HSC73, thereby suppressing MHC class I‐restricted presentation of HSC73‐bound antigenic peptides.