A mimic of phosphorylated prolactin (S179D PRL) inhibits mouse normal mammary HC11 cell proliferation through the upregulation of the vitamin D receptor (VDR) and p21. Here, we investigated whether S179D PRL also inhibited growth of human breast cancer MCF7 cells via VDR and p21. Western blots showed that S179D PRL upregulated VDR and p21 after the cells were incubated with S179D PRL for 3 days. These effects were blocked by the MAP kinase blocker PD98059 (25 μM), indicating that MAPK plays a role in VDR and p21 upregulation. To confirm whether VDR contributes to p21 upregulation, we used two constructs that express luciferase. One (p21 VDRE Luc) has the vitamin D response element (VDRE) in the p21 promoter region; the other (p21 NO-VDRE Luc) does not. The results show that S179D PRL upregulated p21 VDRE Luc activity in p21 VDRE Luc-transfected cells more than in p21 NO-VDRE-transfected cells, indicating that S179D PRL upregulated p21 via VDR. A cell proliferation assay showed that S179D PRL inhibits cell proliferation in a dose-dependent manner.