Substrates, the microbial community, and volatile fatty acids (VFAs) are interacting factors in anaerobic fermentation. In this paper, a microfiltration membrane was installed in a conventional reactor for sludge anaerobic fermentation, and changes in the VFA composition and the microbial community structure after the application were monitored. The membrane rejected much more polysaccharide than protein. VFA production, mainly acetic acid, was greatly enhanced by the membrane separation. The glycolytic species Christensenella minuta was significantly enriched. The explanation was inferred to be the higher retention rate of polysaccharide compared with proteins by the microfiltration. However, surprisingly, another polysaccharide using genus, Parabacteroides, did not benefit from the application of the membrane separation, suggesting more complicated mechanisms might be involved.