Androgens have been recognized to be primary causative agents of prostate cancer. Following binding to the androgen receptor (AR), androgens serve important roles in the carcinogenesis of prostate cancers. ARs serve an important role during all stages of prostate cancer, and inhibiting their function may help to slow prostate cancer growth. In the present study, the AR gene was targeted in androgen?positive prostate cancer cells using the clustered regularly interspaced short palindromic repeats?associated protein (CRISPR/Cas) system. A total of three different single?guide RNAs (sgRNAs) were designed according to the three different target sites in the AR gene. The optimal sgRNA with a specific target effect was effectively screened to cleave the AR gene in androgen?positive prostate cancer cell lines, and to suppress the growth of androgen?sensitive prostate cancer in?vitro. The AR?sgRNA?guided CRISPR/Cas system was able to disrupt the AR at specific sites and inhibit the growth of androgen?sensitive prostate cancer cells; further studies demonstrated that the decreased cell proliferation was due to cellular apoptosis. The results of the present study suggested that the CRISPR/Cas system may be a useful therapeutic strategy for the treatment of prostate cancer.