Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression andcell cycle progression. The active mutant IPP5 (8-60hIPP5m), the latest member of the inhibitory molecules forPP1, has been shown to inhibit the growth of human cervix carcinoma cells (HeLa). In order to elucidate theunderlying mechanisms, the present study assessed overexpression of 8-60hIPP5m in HeLa cells. Flow cytometricand biochemical analyses showed that overexpression of 8-60hIPP5m induced G2/M-phase arrest, which wasaccompanied by the upregulation of cyclin B1 and phosphorylation of G2/M-phase proteins ATM, p53, p21cip1/waf1and Cdc2, suggesting that 8-60hIPP5m induces G2/M arrest through activation of the ATM/p53/p21cip1/waf1/Cdc2/cyclin B1 pathways. We further showed that overexpression of 8-60hIPP5m led to delayed nuclear translocationof cyclin B1. 8-60hIPP5m also could translocate to the nucleus in G2/M phase and interact with pp1α and Cdc2as demonstrated by co-precipitation assay. Taken together, our data demonstrate a novel role for 8-60hIPP5min regulation of cell cycle in HeLa cells, possibly contributing to the development of new therapeutic strategiesfor cervix carcinoma.