We show that MLCK (myosin light chain kinase) plays a key role in cell cycle progression of hepatocytes: either chemical inhibitor ML7 or RNA interference led to blockade of cyclin D1 expression and DNA replication, providing evidence that MLCK regulated S phase entry. Conversely, inhibition of RhoK by specific inhibitor Y27632 or RhoK dominant-negative vector did not influence progression in late G1 and S phase entry. Inhibition of either MLCK or RhoK did not block ERK1/2 phosphorylation, whereas MLCK regulated ERK2-dependent p70S6K activation. In addition, DNA synthesis was reduced in hepatocytes treated with p70S6K siRNA, demonstrating the key role played by the kinase in S phase entry. Interestingly, after the G1/S transition, DNA replication in S phase was no longer dependent on MLCK activity. We strengthened this result by ex vivo experiments and evidenced an MLCK-dependent window in late G1 phase of regenerating liver after two-thirds partial hepatectomy. In conclusion, our results underline an MLCK-dependent restriction point in G1/S transition, occurring downstream of ERK2 through the regulation of p70S6K activation, and highlighting a new signaling pathway critical for hepatocyte proliferation.