Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I α-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc3Man9GlcNAc2 as an essential step in this pathway. However, the extent of mannose removal has not been determined. We show here that glycoproteins subject to endoplasmic reticulum-associated degradation undergo reglucosylation, deglucosylation, and mannose trimming to yield Man6GlcNAc2 and Man 5GlcNAc2. These structures lack the mannose residue that is the acceptor of glucose transferred by UDP-Glc:glycoprotein glucosyltransferase. This could serve as a mechanism for removal of the glycoproteins from folding attempts catalyzed by cycles of reglucosylation and calnexin/calreticulin binding and result in targeting of these molecules for proteasomal degradation.