AbstractLeukemia is a group of cancer caused by the abnormal proliferation and differentiation of hematopoietic stem cells. Efforts geared toward effective therapeutic strategies with minimal side effects are underway. Peptides derived from natural resources have recently gained special attention as alternative chemotherapeutic agents due to their minimal adverse effects. In the present study, the aim was to isolate peptides from garlic (Allium sativum) and investigate their anticancer activity against leukemic cell lines. The protein extract of A. sativum was pepsin‐digested to obtain protein hydrolysate followed by sequential purification methods. A novel anticancer peptide, VKLRSLLCS (VS‐9), was identified and characterized by mass spectrometric analysis. The peptide was demonstrated to significantly inhibit the cell proliferation of MOLT‐4 and K562 leukemic cell lines while exhibiting minimal inhibition against normal PBMC. Particularly, VS‐9 could induce apoptosis and upregulate mRNA levels of caspase 3, caspase 8, caspase 9, and Bax while downregulating Bcl‐2, Bcl‐xL, and Bcl‐w. Molecular docking of VS‐9 with the anti‐apoptotic Bcl‐2 protein family suggested that VS‐9 could bind the binding groove of the BH3 domain on target proteins. Protein–peptide interaction analysis by affinity chromatography and LC‐MS/MS further showed that VS‐9 could bind Bcl‐2 proteins. Results suggest VS‐9 as a potential garlic‐derived novel anticancer peptide possessing apoptosis‐inducing properties against leukemic cell lines via anti‐apoptotic Bcl‐2 protein family.