Given that exosomes mediate intercellular communication by delivering cellular components to recipient cells or tissue, they have the potential to be engineered to deliver therapeutic payloads. However, the regulatory mechanism of exosome secretion is poorly understood. In addition, mitochondrial components have been found in exosomes, suggesting communication between mitochondria and exosomes. However, the molecular mechanism of the mitochondria and vesicle interaction remains unclear. Here, we showed that mitochondrial thioredoxin 2 (TRX2) decreased exosome concentrations and inhibited HCT116 cell migration. Coimmunoprecipitation/mass spectrometry (Co-IP/MS) showed that TRX2 interacted with Rab35. TRX2 and Rab35 bound to each other at their N-terminal motifs and colocalized on mitochondria. Furthermore, TRX2 induced Rab35 degradation, resulting in impaired exosome secretion. Additionally, Rab35 mediated the suppressive effects of TRX2 on cell migration, and TRX2 suppressed cell migration through exosomes. Taken together, this study first found an interaction between TRX2 and Rab35. These results revealed a new role for TRX2 in the regulation of exosome secretion and cell migration and explained the upstream regulatory mechanism of Rab35. Furthermore, these findings also provide new molecular evidence for communication between mitochondria and vesicles.