Although diverse immunomodulatory reactions of probiotic bacteria have been reported, this effect viaBacillus subtilisnatto remains unclear, despite its long consumption history in Japan and usage in Natto production. Hence, we performed a comparative analysis of the immunomodulatory activities of 23 types ofB. subtilisnatto isolated from Natto products to elucidate the key active components. Among the isolated 23 strains, the supernatant fromB. subtilisstrain 1 fermented medium showed the highest induction of anti-inflammatory IL-10 and pro-inflammatory IL-12 in THP-1 dendritic cells (THP-1 DC) after co-incubation. We isolated the active component from strain 1 cultured medium and employed DEAE-Sepharose chromatography with 0.5 M NaCl elution for fractionation. IL-10-inducing activity was specific to an approximately 60 kDa protein, GroEL, which was identified as a chaperone protein and was significantly reduced with anti-GroEL antibody. Differential expression analysis of strains 1 and 15, which had the lowest cytokine-producing activity, showed a higher expression of various genes involved in chaperones and sporulation in strain 1. Furthermore, GroEL production was induced in spore-forming medium. The present study is the first to show that the chaperone protein GroEL, secreted byB. subtilisnatto during sporulation, plays a crucial role in IL-10 and IL-12 production in THP-1 DC.