Human Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) which encodes survivin exhibits multiple biological activities, such as cell proliferation and apoptosis. Survivin is overexpressed in numerous malignant diseases including acute myeloid leukemia (AML). Recent studies have shown that the CRISPR/Cas9 nuclease-mediated gene-editing systems are suitable approach's for editing or knocking out various genes including oncogenes. We used CRISPR-Cas9 to knockout the BIRC5 in the human leukemic cell line, HL60, and KG1, and these cell lines were transfected with either the Cas9- and three sgRNAs expressing plasmids or negative control (scramble) using Lipofectamine 3000. The efficacy of the transfection was determined by quantitative reverse transcription-polymerase chain (RT-qPCR) and surveyor mutation assays. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. We have successfully knocked out the BIRC5 gene in these leukemic cells and observed that the BIRC5-knocked out cells by CRISPR/Cas9 showed a significant decrease (30 folds) of survivin at mRNA levels. Moreover, cell death and apoptosis were significantly induced in BIRC5-CRISPR/Cas9-transfected cells compared to the scramble vector. We demonstrated for the first time that targeting BIRC5 by CRISPR/Cas9 technology is a suitable approach for the induction of apoptosis in leukemic cells. However, further studies targeting this gene in primary leukemic cells are required. © 2019 Narimani et al.