In yeast, animals and other organisms, storage lipids are maintained in the cytoplasm in specialized organelles called lipid droplets (LDs). In the last decade, proteomic and genetic studies of this compartment have shown that LDs appear as a complex dynamic organelle with a role in metabolism control and cell signaling. LDs have received a lot of attention as their abnormal dynamics is associated with several metabolic diseases (obesity, diabetes, atherosclerosis and myopathies). In the context of green chemistry, LDs are also promising sources of neutral lipids for the development of derived products, such as biofuels and alternative molecules for food, medicine and cosmetics. Previous studies highlighted the role of trafficking pathways in LD dynamics. This was reinforced by LD proteomics which suggested that a significant number of trafficking proteins are associated with this organelle, including several Rab GTPases. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7 mutants and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Mutants of the homotypic fusion and vacuole protein sorting (HOPS) complex showed similar phenotypes. Using co-immunoprecipitation, we isolated several Ypt7p partners including, Vma13p, the H subunit of the vacuolar (H+) ATPase. Deletion of the VMA13 gene also increased the cell fatty acid content and LD number. We showed that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and is impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.