Bacteroides species are the most abundant Gram-negative bacteria of the human colonic microbiota. These endogenous organisms are unique in that they synthesize an extensive number of phase-variable surface polysaccharides. Pathogenic bacteria phase vary expression of surface molecules for immune evasion, but the importance of the synthesis of multiple phase-variable polysaccharides to these commensal bacteria is unknown. We previously showed that a Bacteroides fragilis mutant unable to synthesize 4 of the 8 capsular polysaccharides and unable to glycosylate proteins properly is rapidly outcompeted by the wild-type strain for colonization of the gnotobiotic mouse intestine. In the present study, we constructed mutants defective only in capsule polysaccharide synthesis to define better the importance of these surface molecules to intestinal colonization. We discovered a key enzymatic activity required for synthesis of 7 of the 8 capsular polysaccharides. Deletion of its gene resulted in the first B. fragilis mutant able to synthesize only one phase-variable polysaccharide, and further mutation resulted in a stable acapsular mutant. We show that the acapsular mutant is rapidly outcompeted, but synthesis of a single polysaccharide is sufficient for the organism to colonize the gnotobiotic intestine competitively. These data demonstrate that initial colonization of the gnotobiotic mouse intestine by B. fragilis requires that the organism synthesize only a single polysaccharide and suggest that the synthesis of multiple phase-variable polysaccharides is important for the bacteria's long-term maintenance in the normally complex and competitive ecosystem.