Secreted proteins of eukaryotes are decorated with branched carbohydrate oligomers called glycans. This fact is only starting to be considered for in silico investigations of protein dynamics. Using all-atom molecular dynamics (MD) simulations and Markov state modeling (MSM), we unveil the influence of glycans on the conformational flexibility of the multidomain protein disulfide isomerase (PDI), which is a ubiquitous chaperone in the endoplasmic reticulum (ER). Yeast PDI (yPDI) from Saccharomyces cerevisiae is glycosylated at asparagine side chains and the knowledge of its five modified sites enables a realistic computational modeling. We compare simulations of glycosylated and unglycosylated yPDI and find that the presence of glycan-glycan and glycan-protein interactions influences the flexibility of PDI in different ways. For example, glycosylation reduces interdomain interactions, shifting the conformational ensemble toward more open, extended structures. In addition, we compare our results on yPDI with structural information of homologous proteins such as human PDI (hPDI), which is natively unglycosylated. Interestingly, hPDI lacks a surface recess that is present in yPDI. We find that glycosylation of yPDI facilitates its catalytic site to reach close to this surface recess. Hence, this might point to a possible functional relevance of glycosylation in yeast to act on substrates, while glycosylation seems redundant for the human homologous protein. We conclude that glycosylation is fundamental for protein dynamics, making it a necessity for a truthful representation of the flexibility and function in in silico studies of glycoproteins.