Background Transforming growth factor (TGF) beta signalling pathway plays a crucial role in liver regeneration following partial hepatectomy in mice. Evidence demonstrated that beta-2 Spectrin is involved in TGF beta/Smad signalling pathway as a Smad3/4 adaptor protein. Aim The aim of this study was to explore the role of beta-2 Spectrin in hepatocyte proliferation. Methods beta-2 Spectrin expression was evaluated in mice receiving partial hepatectomy. The effect of siRNA against beta-2 Spectrin on hepatocyte proliferation was determined. The interaction between TGF beta/Smad and PI3K/Akt signalling was investigated. Results Hepatic beta-2 Spectrin decreased dramatically 2days after 70% hepatectomy in mice. In AML-12 cells, hepatocyte proliferation was inhibited after the stimulation of TGF beta 1 and a reduction in beta-2 Spectrin mediated by siRNA resulted in increase in proliferative response. Confocal results revealed that beta-2 Spectrin represented a key regulator in TGF beta/Smad signalling through controlling Smad3/4 subcellular localization. Moreover, Alternation of Akt phosphorylation led to the change in subcellular localization of Smad2, 3, 4 and beta-2 Spectrin, A reduction in Smad2, 3 and 4 mediated by siRNA resulted in the induction of pAkt expression. Conclusions These findings reveal that beta-2 Spectrin plays a crucial role in hepatocyte proliferation, which contributes to liver regeneration following hepatectomy in mice. In addition, PI3K/Akt is involved in TGF beta/Smad signalling pathway through the interaction with Smad proteins and beta-2 Spectrin.