α-1,3-Glucan is one of the main polysaccharides in the cell wall of Aspergillus nidulans. We previously revealed that it plays a role in hyphal aggregation in liquid culture, and that its molecular mass (MM) in an agsA-overexpressing (agsA^OE) strain was larger than that in an agsB-overexpressing (agsB^OE) strain. The mechanism that regulates its MM is poorly understood. Although the gene amyD, which encodes glycosylphosphatidylinositol (GPI)-anchored α-amylase (AmyD), is involved in the biosynthesis of α-1,3-glucan in A. nidulans, how it regulates this biosynthesis remains unclear. Here we constructed strains with disrupted amyD (ΔamyD) or overexpressed amyD (amyD^OE) in the genetic background of the ABPU1 (wild-type), agsA^OE, or agsB^OE strain, and characterized the chemical structure of α-1,3-glucans in the cell wall of each strain, focusing on their MM. The MM of α-1,3-glucan from the agsB^OEamyD^OE strain was smaller than that in the parental agsB^OE strain. In addition, the MM of α-1,3-glucan from the agsA^OE ΔamyD strain was greater than that in the agsA^OE strain. These results suggest that AmyD is involved in decreasing the MM of α-1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions.