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The cyclin-dependent kinase inhibitor p21 plays a critical role in regulating cell cycle and cell proliferation. We previously cloned the dog p21 gene and found that unlike human p21, dog p21 is expressed as 2 isoforms due to the proline-directed phosphorylation at serine 123 (S123). Here, we identified that PPM1D, also called Wip1 and a Mg(2+)-dependent phosphatase, dephosphorylates dog p21 protein at serine 123. Specifically, we showed that the level of S123-phosphorylated dog p21 is increased by a PPM1D inhibitor in a dose-dependent manner. We also showed that over-expression of PPM1D decreases, whereas knockdown of PPM1D increases, the level of S123-phosphorylated dog p21 regardless of p53. Additionally, in vitro phosphatase assay was performed and showed that phosphorylated S123 in dog p21 is dephosphorylated by recombinant rPPM1D, which contains the catalytic domain of human PPM1D (residue 1-420), but not by the phosphatase dead rPPM1D (D314A). Furthermore, dephosphorylation of S123 by rPPM1D can be abrogated by PPM1D inhibitor or by withdrawal of Mg(2+). Finally, we showed that upon PPM1D inhibition, the level of S123-phosphorylated dog p21 was increased, concomitantly with decreased expression of cyclin A, cyclin B, Rb, and PCNA. Together, our results indicate that PPM1D functions as a phosphatase of dog p21 at serine 123 and plays a role in cell cycle control via p21.