SUMOylation is a post-translational modification involved in diverse cellular processes in eukaryotes. Previous studies demonstrated that CRISPRi-mediated knockdown (KD) of the Giardia lamblia SUMO-conjugating enzyme Ubc9 (GlUbc9, GL50803_24068) impairs cell cycle progression and maintenance of normal cell morphology in G. lamblia. Here, we investigated the role of SUMOylation during Giardia encystation. GlUbc9 was continuously expressed throughout in vitro encystation, and GlUbc9 KD cells exhibited significantly reduced cyst formation efficiency compared with control cells. Transmission electron microscopy analysis revealed that most cyst-like forms in GlUbc9 KD cells at 48 h post-induction lacked a recognizable cyst wall, resulting in naked cysts. The major components of the G. lamblia cyst wall are cyst wall proteins (GlCWPs) and β-1,3-N-acetylgalactosamine homopolymers, which are transported to the cell surface via encystation-specific vesicles (ESVs) and encystation-specific carbohydrate-positive vesicles, respectively. Neither the expression levels of GlCWP1 and GlCWP2 nor the proteolytic processing of GlCWP2 and assembly of GlCWP1-GlCWP2-N were substantially altered in GlUbc9 KD cells. However, ESVs accumulated in GlUbc9 KD encysting cells, suggesting delayed ESV maturation and/or altered ESV dynamics. In contrast, secretion of N-acetylgalactosamine-containing glycoconjugates and encystation-specific carbohydrate-positive vesicle formation were not markedly altered. Taken together, these findings indicate that SUMOylation is required for Giardia encystation and suggest that its role is associated with the timely maturation and/or dynamics of ESVs rather than with cyst wall protein expression or processing.