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Most eukaryotic cells are polarized. Common toolbox regulating cell polarization includes Rho gua-nosine triphosphatases (GTPases), in which spatio-temporal activation is regulated by a plethora of reg-ulators. Rho of plants (ROPs) are the only Rho GTPases in plants. Although vesicular trafficking was hinted in the regulation of ROPs, it was unclear where vesicle-carried ROP starts, whether it is dynamically regulated, and which components participate in vesicle-mediated ROP targeting. In addition, although vesicle trafficking and guanine nucleotide inhibitor (GDI) pathways in Rho signaling have been extensively studied in yeast, it is unknown whether the two pathways interplay. Unclear are also cellular and developmental con-sequences of their interaction in multicellular organisms. Here, we show that the dynamic targeting of ROP through vesicles requires coat protein complex Ⅱ and ADP-ribosylation factor 1-mediated post-Golgi trafficking. Trafficking of vesicle-carried ROPs between the plasma membrane and the trans-Golgi network is mediated through adaptor protein 1 and sterol-mediated endocytosis. Finally, we show that GDI and vesicle trafficking synergistically regulate cell polar-ization and ROP targeting, suggesting that the estab-lishment and maintenance of cell polarity is regulated by an evolutionarily conserved mechanism.