The aim of the present study was to use the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‑associated (Cas) 9‑mediated gene knockout technology for the rapid classification of the differential function of micro (mi)RNAs screened using miRNA expression profiling by microarray. The rational design of single guide RNAs for the CRISPR/Cas9 system was verified to function in human LNCaP cells with rapid and efficient target gene editing. miRNA (miR)‑205, miR‑221, miR‑222, miR‑30c, miR‑224, miR‑455‑3p, miR‑23b and miR‑505 were downregulated in patients with prostate cancer (PCa) and were experimentally validated to function as tumor suppressors in prostate cancer cells, affecting tumor proliferation, invasion and aerobic glycolysis. In addition, the data of the present study suggested that miR‑663a and mfiR‑1225‑5p were upregulated in prostate cancer tissues and cell proliferation of miR‑663a and miR‑1225‑5p knockout PCa cells was significantly lower compared with miR‑NC cells. Furthermore, knockout of miR‑1225‑5p and miR‑663a significantly decreased the lactate production in LNCaP cells in vitro. In conclusion, the present study offered a simple and efficient method for rapidly classifying miRNA function by applying CRISPR/Cas9 in LNCaP cells. The present study suggested, for the first time to the best of the authors' knowledge, that the aberrant expression of miR‑663a and miR‑1225‑5p may be involved with the progression of prostate cancer, implying their potential as candidate markers for this type of cancer. However, the precise role of miR‑663a and miR‑1225‑5p in accelerating the development of prostate cancer and promoting tumor progression remains to be elucidated.