Mitochondria have generated the bulk of ATP to fuel cellular activities, including membrane trafficking, since the beginning of eukaryogenesis. How inhibition of mitochondrial energy production will affect the form and function of the endomembrane system and whether such changes are specific in today's cells remain unclear. Here, we treated Arabidopsis thaliana with antimycin A (AA), a potent inhibitor of the mitochondrial electron transport chain (mETC), as well as other mETC inhibitors and an uncoupler. We investigated the effects of AA on different endomembrane organelles connected by vesicle trafficking via anterograde and retrograde routes that heavily rely on ATP and GTP provision for SNARE and RAB/GEF function, respectively, in root cells. Similar to previous reports, AA inhibited root growth mainly by shortening the elongation zone (EZ) in an energy- and auxin-dependent way. We found that PIN-FORMED 2 (PIN2) and REQUIRES HIGH BORON 1 (BOR1), key proteins for EZ establishment and cell expansion, undergo accelerated endocytosis and accumulate at enlarged multivesicular bodies (MVBs) after AA treatment. Such accumulation is consistent with the observation that the central vacuole becomes fragmented and spherical and that the Arabidopsis Rab7 homolog RABG3f, a master regulator of MVB and vacuolar function, localizes to the tonoplast, likely in a GTP-bound form. We further examined organelles and vesicle populations along the secretory pathway and found that the Golgi apparatus-in particular, the endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-cannot be maintained when mETC is inhibited. Our findings reveal the importance and specific impact of mitochondrial energy production on endomembrane homeostasis.