Abstract Liver regeneration, following partial hepatectomy (PHx), occurs through precisely controlled and synchronized cell proliferation, in which quiescent hepatocytes undergo one to two rounds of replication, with restoration of liver mass and function. We previously demonstrated that loss of the Smad3/4 adaptor protein β-2 spectrin (β2SP) is associated with faster entry into S phase, and hepatocellular cancer formation. These observations led us to further pursue the role of β2SP in cell cycle progression in vivo . Liver regeneration studies with PHx in β2SP+/− mice reveal a surprising and significant decrease in liver/body weight ratio at 48 hours after PHx in β2SP+/− mice in comparison to wildtype mice. At 48 hours after PHx we also observe decreased levels of cyclin E (2.4-fold, P < 0.05), Cdk1 (7.2-fold, P < 0.05), cyclin A, pRb (Ser249/Thr252), proliferative cell nuclear antigen (PCNA), cyclin D1 with elevated levels of pCdk1 (Thr14) (3.6-fold, P < 0.05). Strikingly, at 24 hours elevated levels of p53 (4-fold, P < 0.05), phospho-p53 (ser15 and ser20), and p21 (200-fold, P < 0.05) persisting to 48 hours after PHx further correlated with raised expression of the DNA damage markers pChk2 (Thr68) and γH2AX (S139). However, compromised cell cycle progression with loss of β2SP is not rescued by inhibiting p53 function, and that G2/M phase arrest observed is independent and upstream of p53. Conclusion : β2SP deficiency results in dysfunctional hepatocyte cell cycle progression and delayed liver regeneration at 48 hours after PHx, which is p53-independent. β2SP loss may increase susceptibility to DNA damage, impair cell cycle progression, and ultimately lead to hepatocellular cancer. (Hepatology 2011;)