Objective In order to establish a rhesus monkey model of p53 gene silencing, firstly we screened and determined the effective silencing targets of p53 gene at the cellular level in rhesus monkey.Methods The expression of p53 gene was detected in COS-7 cells (derived from the kidney of the African Green Monkey, Cercopithecus aethiops).Three small hairpin RNA (shRNA) sequences targeting rhesus monkey p53 gene were designed, analysed by bioinformatics, and inserted into lentivirus-based gene silencing constructs FUGW-TDT. The plasmids of p53-RNAi and control vector were transfected into the COS-7 cells, respectively.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western blot assay. Results p53 gene expression was detected in COS-7 cells.Bioinformatics analysis showed that three gene-silencing sequences were screened which lied in the open reading frame (ORF) region and targeted 238-258bp, 681-701bp, 169-189bp of the rhesus monkey p53 mRNA.At 48 hrs after transfection of the three silencing constructs, p53 mRNA was suppressed by(87.17±4.03)%, (72.62±4.11)% and(76.22±0.98)%, and p53 protein was suppressed by (84.44±2.18)%, (71.04±1.18)% and (74.17±0.95)%, respectively. Conclusions We obtained three effective target sequences showing high efficiency in p53silencing, which can be used in further studies on gene silencing in rhesus monkey.