Gamma-interferon-inducible lysosomal thiolreductase (GILT) promotes major histocompatibility complex (MHC) class II-restricted presentation of exogenous antigens containing disulfide bonds. Here, we show that GILT also facilitates MHC class I-restricted recognition of such antigens by CD8(+) T cells, or cross-presentation. GILT is essential for cross-presentation of a CD8(+) T cell epitope of glycoprotein B (gB) from herpes simplex virus 1 (HSV-1) but not for its presentation by infected cells. Initiation of the gB-specific CD8(+) T cell response during HSV-1 infection, or cross-priming, is highly GILT-dependent, as is initiation of the response to the envelope glycoproteins of influenza A virus. Efficient cross-presentation of disulfide-rich antigens requires a complex pathway involving GILT-mediated reduction, unfolding, and partial proteolysis, followed by translocation into the cytosol for proteasomal processing.