The thiol-oxidoreductase ERp57 is an integral component of the peptide-loading complex of the major histocompatibility complex (MHC) class I pathway, but its function is unknown. To investigate its function in antigen presentation, we generated ERp57-deficient mice. Death in utero caused by ubiquitous ERp57 deletion was prevented by specific deletion in the B cell compartment. We demonstrate that ERp57 was central for recruitment of MHC class I molecules into the loading complex. In ERp57-deficient cells, we found short-lived interaction of MHC class I molecules with the loading complex. Thus, in the steady state, very few MHC class I molecules were present in the loading complex. Surface H-2K(b)-peptide expression and stability were reduced, and presentation of a model antigen was decreased. Our results indicate that ERp57 does not influence the redox state of MHC class I molecules but is an essential structural component required for stable assembly of the peptide-loading complex.