HMGN5 is a typical member of the HMGN (high mobility group nucleosome-binding protein) family whichmay function as a nucleosomal binding and transcriptional activating protein. Overexpression of HMGN5 hasbeen observed in several human tumors but its role in tumorigenesis has not been fully clarified. To investigateits significance for human lung cancer progression, we successfully constructed a shRNA expression lentiviralvector in which sense and antisense sequences targeting the human HMGN5 were linked with a 9-nucleotide loop.Inhibitory effects of siRNA on endogenous HMGN5 gene expression and protein synthesis were demonstratedvia real-time RT-PCR and western blotting. We found HMGN5 silencing to significantly inhibit A549 andH1299 cell proliferation assessed by MTT, BrdU incorporation and colony formation assays. Furthermore, flowcytometry analysis showed that specific knockdown of HMGN5 slowed down the cell cycle at the G0/G1 phaseand decreased the populations of A549 and H1299 cells at the S and G2/M phases. Taken together, these resultssuggest that HMGN5 is directly involved in regulation cell proliferation in A549 and H1299 cells by influencingsignaling pathways involved in cell cycle progression. Thus, our finding suggests that targeting HMGN5 maybe an effective strategy for human lung cancer treatment.