The molecular chaperone HSP60 is a chaperonin homolog of GroEL. We had previously shown that the immunosuppressant mizoribine is bound directly to HSP60 and inhibited its chaperone activity. However, the inhibitory mechanisms of HSP60 by mizoribine have not yet been fully understood. In the present study, we investigated the influence of mizoribine on a folding cycle of HSP60 and co-chaperone HSP10. Our results showed that mizoribine inhibited the folding cycle of HSP60/HSP10. The ATPase activity of HSP60/HSP10 was decreased in the presence of mizoribine and the dissociation of HSP10 from HSP-60 was also decreased by mizoribine. The same functions of GroEL and/or GroES were slightly affected by mizoribine. Based on our findings, we discuss the inhibitory mechanisms of HSP60 by mizoribine.