Objective  To investigate the effects of knockdown the expression of the voltage-gated calcium channel α2δ1 subunit on the stem cell-like traits in the tumor-initiating cell enriched Hep-12 hepatocellular carcinoma cell line through clustered regularly interspaced short palindromic repeats/-associated protein 9(CRISPR/Cas9) system.   Methods  Three pairs of single guide RNA (sgRNA) targeting α2δ1 were constructed into the lenti CRISPRv2-puro vector using standard DNA recombinant technique, and their cleavage activities were verified in vitro. The lenti CRISPRv2-puro vector containing the sgRNA sequence was packaged into lentivirus in 293FT cells using the 3rd generation packing system. The viruses were then used to infect Hep-12 cells which were subsequently selected with puromycin. The expression of α2δ1 and stemness related genes were detected by Western blotting, and the in vitro self-renewal capacity was measured by spheroid formation assay.   Results  The designed sgRNAs targeting α2δ1 gene were demonstrated to cleave α2δ1 DNA fragment efficiently in vitro, and the lenti CRISPRv2-puro vectors harboring the corresponding sgRNA sequences were successfully constructed. Compared with that of control cells, the expression of α2δ1 and stem cell associated genes B-cell-specific moloney leukemia virus insertion site 1(BMI1), and Nanog were remarkably suppressed following the infection with the lentivirues harboring sgRNAs against α2δ1 in Hep-12 cells. Furthermore, the in vitro self-renewal ability of Hep-12 cells was retarded significantly as evidenced that the spheroid formation efficiency in serum free medium of these cells reduced dramatically with knockdown of α2δ1.   Conclusion  The voltage-gated calcium channel α2δ1 is essential for the maintanence of the self-renewal properties of the tumor-intiating cell enriched hepatocellular carcinoma Hep12 cells.