Updated project metadata. Accumulating studies have shown that mitochondria-lipid droplet (LD) interaction is crucial for maintaining the homeostasis of lipid metabolism in the liver. However, the proteins involved in mitochondria-LD interaction and the functioning mechanism remain largely elusive. Here, we applied a systematic approach to characterize the proteome of mitochondria-LD contacts under normal or starvation condition by combining the bimolecular fluorescence complementary assay and a proximity labeling strategy. We first established a reporter of mitochondria-LD contacts in HepG2 cells and demonstrated that mitochondria-LD contacts are pretty sensitive to nutritional stresses. Furthermore, we uncovered 60 proteins are up-or down-regulated at contact sites upon starvation. Finally, we validated that SQLE translocates from endoplasmic reticulum to LDs and accumulates at mitochondria-LD contact sites upon starvation so as to utilize local ATP for cholesterol synthesis. This work also provides a versatile tool to profile the proteomes of any other inter-organelle membrane contacts in living cells.