We have identified insertional mutants of mannosidase 1A and xylosyltransferase 1A in Chlamydomonas reinhardtii. Mass spectrometric analyses of intact N-glycopeptides revealed that disruption of the genes altered the N-glycan composition of the alga (in single as well as double mutant). In contrast to wildtype, methylated hexoses and terminal xylose were nearly absent in N-glycopeptides from the Man1A mutant strain whereas the XylT1A mutant showed a lack of core xylose and excessively trimmed N-glycans. The double mutant did not show this excessive trimming, thus Man1A-dependent trimming can be concluded in C. reinhardtii. Furthermore, these data indicate that methylation of hexoses is tightly interlinked with Man1A function, pointing to an enzymatic cascade in the N-glycosylation pathway.