Ras homolog enriched in brain (Rheb), a small GTPase, positively regulates the mTORC1 pathway. The GDP-GTP exchange of Rheb has been suggested to be facilitated by translationally controlled tumor protein (TCTP). Here we demonstrate that human TCTP (hTCTP) interacts with human Rheb (hRheb) and accelerates its GDP release in vitro and that hTCTP activates the mTORC1 pathway in vivo. To investigate the underlying mechanism, we built structure models of GDP- and GTP-bound hRheb in complexes with hTCTP and performed molecular dynamics simulations of the models, which predict key residues involved in the interactions and region of hRheb undergoing conformational change during the GDP-GTP exchange. These results are verified with site-directed mutagenesis and in vitro biochemical and in vivo cell biological analyses. Furthermore, a crystal structure of the E12V mutant hTCTP, which lacks the guanine nucleotide exchange factor activity, shows that the deficiency appears to be caused by loss of a salt-bridging interaction with Lys-45 of hRheb. These data collectively provide insights into the molecular mechanisms of how hTCTP interacts with hRheb and activates the mTORC1 pathway.