The small GTPase Rheb binds and activates mTORC1, which plays a pivotal role in diverse cellular physiologies. To increase our understanding of how Rheb regulates mTORC1 signaling, we set out to identify Rheb binding proteins using shotgun proteomics approaches. In this study, we characterized HSP70, one of the identified proteins, as a new Rheb binding protein. The present study showed that Rheb forms a complex with HSP70 in intact cells. Interestingly, the binding of Rheb to mTORC1 was abolished by HSP70. Furthermore, the stability of Rheb is dramatically decreased by HSP70, and this degradation is proteasome-dependent. As a result, Rheb-dependent mTORC1 activation was decreased by HSP70. Taken together, HSP70 dissociates Rheb from mTORC1 and induces proteasome-dependent degradation, leading to the inhibition of mTORC1 signaling. Our findings suggest that HSP70 is a negative regulator of mTORC1 signaling via interaction with Rheb. (C) 2020 Published by Elsevier Inc.