Mixed lineage leukemia 5 (MLL5) encodes a mammalian trithorax group (TrxG) protein located within chromosome band 7q22, which is a frequently deleted region found in acute myeloid malignancies. Trithorax and polycomb (PcG) group proteins are evolutionarily conserved transcriptional regulators that maintain the expression of Homeobox (HOX) genes at the epigenetic level during development. Recently, the emerging roles of TrxG and PcG group proteins in cell cycle regulation have begun to be elucidated. In this study, we demonstrated that the mammalian trxG protein MLL5 is involved in multiple cell cycle regulation. Knockdown of MLL5 by small interfering RNA resulted in the retarded cell growth and attenuated intake of BrdU in multiple tumor and normal diploid cells. The cell cycle arrest induced by knockdown of MLL5 took place at both the G1 and G2/M phases. This growth-inhibitory effect and dual-phase arrest were also found in p53-knockout cell lines, suggesting that the transactivation activity of p53 was dispensable for the MLL5-knockdown-mediated cell cycle arrest. In addition, up-regulation of cyclin-dependent kinase inhibitor p21 and de-phosphorylation of retinoblastoma protein were observed in all cell lines tested regardless of their p53 status. Taken together, our data suggest that silencing of MLL5 leads to up-regulation of p21 and dephosphorylation of pRb, which at least partially contributes to the G1 phase and G2/M phase arrest. These findings provide evidence that MLL5 might be an important cell cycle regulator, participating in cell cycle regulatory network machinery at multiple cell cycle stages.