N-glycosylation is an important protein modification that generally occurs at the Asn residue in an Asn-X-Ser/Thr sequon. Ero1 and its homologs play key roles in catalyzing the oxidative folding in the endoplasmic reticulum (ER). Recently, we found that Arabidopsis (Arabidopsis thaliana) ERO1 and AtERO2 displayed different characteristics in catalyzing oxidative protein folding in the ER. All known Erols are glycosylated proteins, including AtERO1 and AtERO2 that were analyzed when they were transiently translated in mammalian cells. However, the exact N-glycosylation sites on AtERO1 and AtERO2 remains to be determined. In this work, using a plant transient expression system, we identified the N-glycosylation sites on both AtERO1 and AtERO2. We found that AtERO1 has one N-glycosylation site, while AtERO2 contains two, all in the N-X-S/T sequons. Interestingly, we found that Ero1 homologs from human, rice, soybean and Arabidopsis, all have a conserved N-glycosylation site near the inner active site that reduces molecular oxygen and provides the oxidizing equivalents. The identification of N-glycosylation sites on AtERO1/2 proteins will help understand the function of N-glycosylation not only in AtERO1/2, but also in other Ero1 homologs. (C) 2020 Elsevier Inc. All rights reserved.