Cells in biofilms dynamically adapt to surrounding environmental conditions, which alters biofilm architecture. The obligate anaerobic pathogenClostridium perfringensshows different biofilm structures in different temperatures. Here we find that the temperature-regulated production of extracellular polymeric substance (EPS) is necessary for morphological changes in biofilms. We identify BsaA proteins as an EPS matrix necessary for pellicle biofilm formation at lower temperature and find that extracellularly secreted BsaA protein forms filamentous polymers. We show thatsipW-bsaAoperon expression is bimodal, and the EPS-producing population size is increased at a lower temperature. This heterogeneous expression of the EPS gene requires a two-component system. We find that EPS-producing cells cover EPS-nonproducing cells attaching to the bottom surface. In the deletion mutant ofpilA2, encoding a type IV pilin, the EPS gene expression is ON in the whole population. This heterogeneity is further regulated by the cleavage of thepilA2mRNA by RNase Y, causing temperature-responsive EPS expression in biofilms. As temperature is an environmental cue,C. perfringensmay modulate EPS expression to induce morphological changes in biofilm structure as a strategy for adapting to interhost and external environments.