MiR-96-5p is a microRNA consisting of 23 nucleotides that originates from precursor-miR-96 and has been known to be involved in the regulation of neuronal GSH synthesis, the migration of cancer cells, and the differentiation both of embryonic stem cells and bone marrow stem cells. However, not all functions have been clarified for this microRNA. In order to investigate unknown features of miR-96-5p in various cell types in vitro, we developed the miR-96-5p gene knockout (KO) system by using CRISPR/Cas9. We designed one target sequence having 5’-NGG PAM sequence which was common to human and mouse miR-96. The target sequence contains a part of miR-96-5p sequence and a cleavage site for Dicer. The activity of CRISPR/Cas9 plasmid (PX459-miR-96 KO) was detected by the EGFP reporter assay in human embryonic kidney (HEK293) cells. In the analysis of DNA sequence, the mutations were found in the expected site in miR-96 gene after the plasmid was electroporated in HEK293 cells. These results show that PX459-miR-96 KO plasmid successfully knockout the miR-96 gene in a human cell line.