Objective To identify variables which affect the efficacy of gene silencing by lentiviral-mediated RNA interference. Methods Both hypoxia-inducible factor-1α (HIF-1α) and hypoxia-inducible factor-1β (HIF-1β) were chosen as the target genes. Lentiviral vectors harboring shRNA expressing constructs were packaged by BLOCK-iT Lentiviral RNAi Expression System (Invitrogen). Vectors were transduced to Hela, SPCA1 and A549 cells respectively, and mRNA abundance was determined by real time RT-PCR. Results The number of lentiviral particles produced by a 10cm tissue culture plate cells was 6.3×1010. Functional titers of Lenti6-HIF1α while transduced Hela, SPCA1 and A549 cells were 1.8×106TU/ml, 1.2×106TU/ml and 1.75×106TU/ml respectively, and they were 1.76×106TU/ml, 1.21×106TU/ml and 1.79×106TU/ml respectively for Lenti6-HIF1β. Longer period of the vector absorption enhanced virus transduction within 12 hours. And the transient RNAi efficacy was proportional related to the quantity of inoculums. Furthermore, the number of shRNA expression constructs integrated into genome of target cells was specifically associated with RNAi efficacy of stable transduced cells. Conclusion In lentiviral-mediated human tumor RNA interference, the transient gene expression inhibition was determined by cell type, quantity of vectors and absorption time, but the stable gene silencing efficacy was determined by viral gene copies integrated into genome of target cells.