Yeast SIT4 is an essential gene encoding a protein Ser/Thr phosphatase conserved throughout eukaryotic evolution, known as PPV in Drosophila and PP6 in vertebrates. Sit4 promotes transcription of G1 cyclins and a sit4(ts) strain exhibits a G1 arrest at the restrictive temperature. The yeast sit4(ts) was rescued by expression of PPV or a chimeric phosphatase containing the first fifty-three residues of PPV fused to Drosophila PP1. The results suggested that the N terminus of the Sit4/PPV protein exerts a specific function in the yeast cell cycle. Here we tested whether the N terminus of human PP6 exerts specific effects on G1-S progression in human cells. The N terminus of PP6 or PP2A was fused to GFP and the proteins transiently expressed in prostate cancer PC-3 cells. Expression of the PP6 fusion protein was restricted to lower levels than either the PP2A fusion protein or GFP. However, the PP6 fusion protein blocked entry into S phase and increased by >20% the proportion of cells in G1 phase. Expression of the PP6 fusion protein did not significantly change the levels of transcripts for cyclins or ca. eighty other cell cycle genes, but did suppress the levels of cyclin D1 protein in cells in G1 phase and reduce the phosphorylation of RB1 at Ser807/811. Thus, our results provide evidence that PP6 regulates cell cycle progression in human cells at least in part through control of cyclin D1 and the function of PP6 is distinct from its homolog Sit4 in yeast.