The STT3 subunit of the oligosaccharyltransferase complex plays a critical role in the N-glycosylation process. From Arabidopsis thaliana to Homo sapiens, two functional STT3 isoforms have been identified, STT3-A and STT3-B. We report that the last transmembrane (TM) segment of STT3-B corresponds to a topogenic determinant that is sufficient for proper integration and orientation of STT3-B C-terminal domain. Notably, the last TM segment of STT3-A and -B isoforms present major differences in amino acid sequence and predicted 3D structure. We also identified a bipartite nuclear targeting sequence in the C-terminal tail of STT3-B that is absent in STT3-A. The latter sequence is sufficient to induce nucleolar localization of a reporter protein. Our results show that STT3-A and -B display two structural differences that may have a drastic influence on their function and might account for the remarkable evolutionary conservation of the two STT3 paralogs.