beta II-Spectrin (beta 2SP), a Smad3/4 adaptor protein during transforming growth factor (TGF) beta/Smad signal pathway, plays a critical role in suppressing hepatocarcinogenesis. Dedifferentiation is a distinctive feature of cancer progression. Therefore, we investigated whether the disruption of beta 2SP contributed to tumorigenesis of hepatocellular carcinoma (HCC) through the dedifferentiation. Down-regulation of beta 2SP in hepatocytes was observed in cirrhotic liver and HCC. The level of beta 2SP expression was closely associated with the differentiation status of hepatocytes in rat model of hepatocarcinogenesis and clinical specimens. Transgenic expression of beta 2SP in HCC cells promoted the differentiation of HCC cells and suppressed the growth of HCC cells in vitro. Efficient transduction of beta 2SP into liver CSCs resulted in a reduction in colony formation ability, spheroid formation capacity, invasive activity, chemo-resistance properties, tumorigenicity in vivo. In addition, beta 2 spectrin exerted its effect through beta catenin in liver CSCs. In conclusion, beta 2 spectrin repressed the properties of liver CSCs through inducing differentiation; thus, strategies to restore its levels and activities would be a novel strategy for HCC prevention and differentiation therapy