Objective To establish stable HL-60 cell line with stable RYBP gene silencing using lentivirus-mediated RNA interference. Methods Five special shRNAs for RYBP gene were cloned to lentivirus vector.Recombinate lentivirus vectors were packed into lentivirus,which were used to infect HL-60 cells, and took empty vector and non-specific shRNA as control groups. Stable infected cells were selected with puromycin in 8 μg/ml concentration.The expression levels of RYBP were analyzed by Western blot,and confirmed the most effective RYBP shRNA.Then the level of mRNA was analyzed by real-time PCR.Results Stable infected cells were selected by puromycin successfully.Comparing to control groups,the expression of RYBP were reduced at different degrees (P ＜ 0.01). And RYBP shRNA2 took the most silencing effect, the RYBP mRNA was decreased by more than 95 ％ (P ＜ 0.05).Conclusion The shRNA2 targeting RYBP gene can effectively inhibit the expression of RYBP. HL-60 cell line with stable RYBP gene silencing were constructed successfully,which had provided experiment fundament for further studying the function of RYBP.