The low yield as bottleneck problem limits the application of microbial flocculant in water treatment. However, genetic information of microbial flocculant-producing strains can guide the regulation of microbial flocculant production, but it remains unknown. Agrobacterium tumefaciens F2 produced polysaccharide-based microbial flocculants in the fermentation medium but none in Luria Bertani medium; hence, the transcriptome was used to analyze the potentially associated genes with the production of microbial flocculants. Glucose, mannose, rhamnose, and galactose are the main sugar monomers, and genes (manA, glmM, manC, rfbgenes, exogenes, etc.) with changed expression levels related to sugar monomers metabolism potentially participated in the biosynthesis of polysaccharide-based microbial flocculants.exoC,exoP, andmanCwere confirmed to participate in the biosynthesis via constructing the mutants F2-dexoC, F2-dexoP, and F2-dmanC. An exo(F2) gene cluster was annotated due to the high percentage of matches between the genome sequences of strains F2 and C58, andexogenes in their genome sequences showed the similarity of 86 similar to 92%. The hypothetical pathway for the biosynthesis of polysaccharide-based microbial flocculants in strain F2 was proposed, laying the basis for the production yield regulation.