Human hepatocellular carcinoma (HCC) is the most frequent cancer worldwide with a poor prognosis. Tumor-specific pyruvate kinase M2 (PKM2) is essential for cancer metabolism and tumorigenesis. Shikonin, a specific inhibitor of PKM2, but not PKM1, exhibits significant anticancer effect in HCC, and was deemed as a promising drug for cancer therapy. However, shikonin-mediated bypass signaling in HCC remained unclear. Here, we performed forward/reverse stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics to identify the early molecular events controlled by shikonin. We demonstrated for the first time that shikonin could induce the nuclear translocation of PKM2 for recruiting Nrf2, and transcriptionally activated Nrf2 downstream target gene BAG3, therefore increasing protective effect to sustain cell survival. Knockdown of BAG3 by si-RNA significantly potentiated the anticancer effect of shikonin. These findings provided the first evidence of a new noncanonical function of inhibited PKM2 could act as a transcriptional coactivator of Nrf2 in cancer survival, highlight that shikonin in combined with BAG3 inhibitor could be a promising therapeutic strategy for HCC therapy.AJCR Copyright © 2021.