In summary, we (1) provided a structural basis for the subcellular location-dependent effects of ACR on TG2 activity; (2) characterized the tumor-promoting roles of cytoplasmic TG2 in HCC and the therapeutic potential of TG2 inhibition in the selective depletion of liver CSCs; and (3) identified a critical role of TG2 in the regulation of EXT1 expression and the downstream signaling, such as HS biosynthesis, partly through TGF-β1 activation-dependent pathways. This evidence concerns the gene EXT1 and hepatocellular carcinoma.