PITX2C overexpression increased the stemness-related characteristics of HCC in in vitro and in vivo functional assays, while mechanistic studies revealed that PITX2C rather than PITX2A/B could activate TGF-β signaling by regulating the transcription of RALYL. Furthermore, PITX2C was found to regulate and cooperate with key factors related to hepatic differentiation and HCC tumorigenicity. This evidence concerns the gene RALYL and hepatocellular carcinoma.