The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its disfunction is correlated with several significant diseases such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) transiently arrests the EXO70 subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N-terminus and C-terminus of the protein. This study provides not only new insights into the novel features of the exocyst complex but also offers a potentially new target for drugs aimed at addressing human disease.