In this study, 10 ns molecular dynamics simulations were conducted to determine the optimum length and type of O-glycosylation in the linker domain of glucoamylase from Aspergillus awamori. The linear distance between the N- and C-termini of the linker domain fluctuated rapidly between 105 and 126 A without adding sugars onto the Thr and Ser residues. Mannose was found to be more favorable to be O-linked to the Ser and Thr residues than galactose under the realistic biophysical conditions. Averagely 5 mannose residues O-linked to the Thr and Ser residues results in the averaged linear distance of 95 A between the N- and C-termini at pH 4.5 and 330K. Hydrogen bonds between the carbonyl oxygen of the 16 Thr residues in the linker domain and the proton in the +1 mannose contribute to stabilizing the O-glycosylated motif.