The bacterial flagellum is involved in a variety of processes including motility, adherence and immunomodulation. In the Clostridioides difficile strain 630Δerm, the main filamentous component, FliC, is post-translationally modified with an O-linked Type A glycan structure. This modification is essential for flagellar function since motility is seriously impaired in gene mutants with improper biosynthesis of the Type A glycan. The cd0240-cd0244 gene cluster encodes the Type A structure, but the role of each gene, and the corresponding enzymatic activity, has not been fully elucidated. Using quantitative mass spectrometry-based proteomics analyses, we determined the relative abundance of the observed glycan variations of the Type A structure in cd0241, cd0242, cd0243 and cd0244 mutant strains. Our data not only confirm the importance of CD0241, CD0242 and CD0243, but, in contrast to previous data, also show that CD0244 is essential for the biosynthesis of the Type A modification. Combined with additional bio-informatic analyses, we propose a revised model for Type A glycan biosynthesis.