Objective The CRISPR/Cas9 editing system was used to stably knock out the ROCK2 gene in human lung cancer cell lines A549 and H460, and the knockout effect was verified. We preliminarily explored the functional changes at the cellular level of the ROCK2 knockout lung cancer cell line. Methods The Rock-2 CRISPR plasmid and Rock-2 HDR plasmid were used to construct A549 and H460 ROCK2 gene knockout stable cell lines. Western blotting was used to verify the knockout effect, and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression level of ROCK2 gene. Colony formation experiments and MTT method were used to detect the proliferation ability of each cell line. Results The results showed that the stable A549 cell line and H460 cell line with deficient ROCK2 expression was successfully obtained. Compared with the original cell line, the protein expression level in the ROCK2 knockout cell line is completely lost, the mRNA expression level in the cell is significantly reduced, and the proliferative abilities were greatly decreased. Conclusion ROCK2 gene knockout lung cancer cell lines provide an effective tool for further research on the function and mechanism of ROCK2 in lung cancer.