Abstract: To construct SAMHD1 gene knockout in Huh7 cell employing the CRISPR/Cas9 system and investigate the effect of SAMHD1 knockout on the proliferation of human hepatoma cells, three small guide RNAs（sgRNA） targeting SAMHD1 were designed for the functional region of the SAMHD1 gene. The lentiCRISPRv2-SAMHD1-g RNA recombinant plasmid was constructed and sequenced. The lentiviral was packaged in HEK293 T cell lines and the SAMHD1 knockout Huh7 cells were selected and identified by sequencing and Western blot. The colony forming assay was used to analyze hepatoma cells proliferation after knockout of SAMHD1. The sequencing results showed that the lentiCRISPRv2-SAMHD1-g RNA vector was successfully constructed. The SAMHD1 knockout Huh7 cells were verified by Western blot and sequencing results. The results colony forming assay showed that the colony number of Huh7-KO SAMHD1 cells were significantly increased compared with parental cell lines（P＜0.01）. The Huh7 cell that knockout SAMHD1 gene employing CRISPR/Cas9 technology was successfully constructed, and SAMHD1 knockout significantly promoted the proliferation of hepatocellular carcinoma cells.