Human papillomavirus (HPVs) type 18 is a major cause of cervical adenocarcinoma. E6 and E7 viral genes play significant roles in the pathogenesis of cervical carcinoma. Single guide RNA (sgRNA)-directed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system is a highly efficient genome engineering tool. Here, we used dual sgRNA-directed CRISPR/Cas9 to delete the HPV18 E6 and E7 genes simultaneously. We deleted the E6 and E7 genes with a high mutation rate of 81.8% in HeLa cells. Deletion of the E6 and E7 genes restored the p53 and Rb protein levels, induced apoptosis and inhibited the proliferation of HeLa cells. Apoptosis and proliferation of the control-SiHa cell line were not affected. Therefore, dual sgRNA-guided CRISPR is a highly specific method for reversing the malignant phenotype of cervical cancer cells. Deletion of the E6 and E7 genes using CRISPR provides us a new therapy to treat HPV18 infection.