Streptococcus pneumoniae (pneumococcus) produces many capsule types that differ in their abilities to evade host immune recognition. To explain these serotypedependent protective capacities, many studies have investigated capsular thickness or the interaction of the capsule with complement proteins, but the effects of small chemical modifications of the capsule on its function have not been studied. One small chemical modification found frequently among pneumococcal capsules is O-acetylation. Pneumococcal serotype 33A has two membrane-bound O-acetyltransferase genes, wciG and wcjE. A 33A wcjE-deficient variant, 33F, occurs naturally and is increasing in prevalence in the wake of widespread conjugate vaccine use, but no wciG-deficient variants have been reported. To study the biological consequence of the loss of O-acetylation, we created wciG-deficient variants in both serotypes 33A and 33F, which we named 33X1 (ΔwciG) and 33X2 (ΔwciG ΔwcjE). Serotypes 33X1 and 33X2 express novel capsule types based on serological and biochemical analyses. We found that loss of WcjE-mediated O-acetylation appears not to affect cell wall shielding, since serotypes 33A and 33F exhibit comparable nonspecific opsonophagocytic killing, biofilm production, and adhesion to nasopharyngeal cells, though serotype 33F survived short-term drying better than serotype 33A. Loss of WciG-mediated O-acetylation in serotypes 33X1 and 33X2, however, resulted in a phenotype resembling that of nonencapsulated strains: increased cell wall accessibility, increased nonspecific opsonophagocytic killing, enhanced biofilm formation, and increased adhesion to nasopharyngeal cells. We conclude that WciG-mediated, but not WcjE-mediated, O-acetylation is important for producing protective capsules in 33A and that small chemical changes to the capsule can drastically affect its biological properties.