Objective To screen the effective silencing targets of P21 gene at the cellular level in rhesus monkey. Methods To detect the expression of P21 gene in COS-7 cells (derived from the kidney of African green monkey, Cercopithecus aethiops). Four small hairpin RNA (shRNA) sequences targeting rhesus monkey P21 gene were designed and inserted into lentivirus-based gene silencing constructs FUGW-TDT. The vectors were transfected into COS-7 cells respectively. The suppression of P21 mRNA was detected by real-time PCR, and the expression of P21 protein was detected by Western blot assay. Results Four gene-silencing sequences were screened that lied in 541-561 bp, 542-562 bp, 215-239 bp, and 624-648 bp of the rhesus monkey P21 mRNA. Their silencing rate was (91.82±3.21)%, (82.47±2.48)%, (81.31±2.69)% and (87.35±4.59)%, and the protein expression was (11.97±0.70)%, (20.22±0.65)%, (23.21±0.63)% and (14.42±0.86)%, respectively. Conclusions Four effective silencing target sequences are screened at cellular level, which can be used in gene silencing research of rhesus monkeys.