Mtf1 has been characterized as a mitochondrial transcription factor and is shown to regulate mitochondrial transcription. Mtf1 has an additional function as a transcription factor for the nuclear gene srk1 in fission yeast. Hsp60 has been linked to a variety of important cellular functions such as apoptosis and the immune response. It functions mainly as a molecular chaperone that assists correct protein folding in the mitochondrion. Epolactaene tertiary butyl ester (ETB) is an inhibitor of human Hsp60 that can inhibit Hsp60 chaperone activity. In this study, we report that in fission yeast, Mtf1 binds to Hsp60 in vivo and in vitro, ETB inhibits the binding of Mtf1 and Hsp60, and inhibits mitochondrial transcription but not nuclear transcription of srk1. We propose that Hsp60 may act as a molecular chaperone that folds mitochondrial Mtf1 into a functional form and that ETB inhibits this Hsp60 chaperone activity by disrupting Mtf1 binding to Hsp60 and thus inhibits mitochondrial transcription in fission yeast.