The distinctive protein and lipid composition underlies the distinct function of each organelle, regulated by balanced anterograde and retrograde membrane trafficking. The vacuole, the largest plant organelle, is pivotal in various plant functions, with its protein composition tightly regulated by bidirectional trafficking. However, the existence of retrograde transport from the plant vacuole remained unverified. In this study, we demonstrate retrograde trafficking from the vacuole in Arabidopsis. We observed retrieval of VAMP727, a plant-unique vacuolar membrane fusion machinery, from the vacuolar membrane. VAMP727 retrieval is facilitated by SORTING NEXIN proteins, which were independently diversified between plant and non-plant systems. Furthermore, we show that the core retromer complex and SORTING NEXINs act independently in distinct retrograde transport events with specific cargos. Thus, plant cells have elaborated a unique retrieval mechanism from the vacuole, underpinning the neofunctionalization of VAMP727 during plant evolution.