Aim： To identify novel small compound inhibitor of p53 protein. Methods： Mouse embryonic fibroblasts （MEF） and mouse embryonic stem （ES） cells were tested. Cell proliferation rate was determined using a Cell Proliferation Kit. The mRNA and protein levels of p53-related genes were measured using real-time PCR and Western blotting, respectively. Global response in the p53 signaling network was analyzed using Illumina whole-genome expression BeadChips. Results： Treatment of MEF cells with a small molecule 1,4-bis-4-（3-phenoxy-propoxy）-but-2-ynyl-piperazine （G5） at 10 pmol/L for 24 h markedly reduced the mRNA and protein levels of the p53 downstream genes MDM2 and p21. In G5-treated ES cells, a total of 372 differentially expressed genes were identified, and 18 among them were direct downstream genes of p53; 6 out of 9 p53-repressed genes were upregulated, and 5 out of 9 p53-activated genes were downregulated. In both MEF cells and ES cells, treatment of with G5 （10 μmol/L） up to 48 h neither affected the proliferation rate nor caused morphological alterations. Conclusion： G5 inhibits p53 activity and simultaneously preserves the normal growth and proliferation of cells, therefore is a new compound for studies of p53-mediated cell manipulation.