Balamuthia mandrillaris is an opportunistic cyst-producing amoeba that can cause rare, but fatal, Balamuthia amoebic encephalitis (BAE). Cysts are resistant to harsh environmental conditions and many antimicrobial compounds and thus can contribute to BAE recurrence. However, little is known of cyst wall synthesis, cyst wall composition, or how encystment is induced. In this study, we examined the carbohydrate composition of the cyst wall. The major components were mannose (20.9 mol%) and glucose (79.1 mol%), with trace amounts of galactose present in the cyst wall samples analysed. The linkage analysis showed cyst wall carbohydrates with apparently linear and branching saccharides and suggested the presence of cellulose. These components may play an important protective role by creating a permeability barrier around the cyst.