Scutellarin, one of natural flavonoids, is widely and clinically used for treating many diseases in China. Recently, scutellarin has demonstrated a broad spectrum of anti-proliferative activities against multiple cancer cell lines. However, the molecular mechanism of action remains to be investigated. We herein report the design and synthesis of biotinylated scutellareins as probes, which can be applied to discover scutellarein interacting proteins. Finally, we show that scutellarin directly targets pyruvate kinase M2 (PKM2) and inhibits its cytosolic activity to decrease glycolytic metabolism; on the other hand, scutellarin may also participate in regulating cell cycle and apoptotic proteins by activating MEK/ERK/PIN1 signaling pathway to promote the nuclear translocation of PKM2.