Objective: To construct a myosin light chain kinase ( MLCK) knockout hypopharyngeal carcinoma FaDu cell line and discuss the knockout effect of MLCK on apoptosis of FaDu cells. Methods: MLCK knockout FaDu cell lines were constructed by liposome transfection of sgRNA and Cas9 2NLS Nuclease , and the knockout cell lines were identified by DNA sequencing and divided into control group , MLCK KO 1 group and MLCK KO 2 group. The knockout efficiency of MLCK was assessed by RT⁃qPCR and Western blot , and the effect of MLCK knockout on cell cycle and apoptosis was detected by flow cytometry while Western bolt was conducted to detect the effect of MLCK knockout on cell apoptosis. Results: DNA sequencing showed that MLCK base sequences were missing or substituted at the sgRNA sequence recognition , and RT⁃qPCR and Western blot showed lower mRNA and protein levels in MLCK knockout cell lines than those in the control group(P < 0. 000 1) . Furthermore , the flow cytometry assay showed that MLCK knockout did not significantly change the cycle of FaDu cells , but the rate of apoptosis increased (P < 0. 000 1) . Western blot showed that Bax/Bcl⁃2 (P < 0. 000 1) and Cleaved Caspase⁃3/Caspase⁃3 (P = 0. 000 7) ratios raised in the MLCK knockout group. Conclusion The knockout of MLCK induced apoptosis in FaDu cells , but the exact mechanism needs further study.