Bioactive natural products are invaluable sources for drug discovery. Unraveling their molecular targets uncovers the mechanisms of action and provides novel targets for drug development. However, the current approaches for target identification fall short in terms of efficiency, due to the extensive list of candidates and limited functional clues. Here we pioneer a strategy that integrates thermal proteome profiling and thermal proximity co-aggregation (TPP-TPCA) for high-efficient target identification. By linking functional targets to downstream perturbed protein complexes, this strategy enables a functional validation of candidate targets. For the first time, we applied this strategy to pinpoint the target of a natural compound veratramine (VAM) with anti-proliferation properties. Notably, the TPP identifies ATP6V1C1 as a candidate target of VAM, while TPCA reveals the dissociation of vacuolar (V)-ATPase. By directly binding to ATP6V1C1, VAM inhibits V-ATPase catalytic activity and lysosomal acidification, ultimately disrupting the autophagic-lysosomal pathway essential for cancer cell survival. Bioinformatics analysis reveals that ATP6V1C1 expression is upregulated in a variety of tumors and serves as a hub gene in breast cancer. Overall, this work presents an efficient strategy for target identification, demonstrating its successful application in identifying ATP6V1C1 as a promising target for cancer treatment.© The author(s).