Durian (Durio zibethinusL.) grows widely in Southeast Asia. The pulp of the durian fruit contains carbohydrates, proteins, lipids, fibers, various vitamins, minerals, and fatty acids. This study was carried out to elucidate the anticancer mechanism of action of the methanolic extract of the fruit ofDurio zibethinus(D. zibethinus) on human leukemia (HL-60) cells. The methanolic extract ofD. zibethinusfruits exhibited its anticancer effect on HL-60 cells by inducing DNA damage and apoptosis. The DNA damage was confirmed by comet and DNA fragmentation assays. The methanolic extract ofD. zibethinusfruits has been shown to cause cell cycle arrest in HL-60 cells during the S phase and G2/M phase. Additionally, the methanolic extract caused induction of the apoptotic pathway in the HL-60 cell line. This was confirmed by increased expression in pro-apoptotic proteins, viz., Bax protein expression, and a substantial reduction (p< 0.001) in anti-apoptotic proteins, viz., Bcl-2 and Bcl-xL expressions. Therefore, this study confirms that the methanolic extract ofD. zibethinusexerts its anticancer effects on the HL-60 cell line, causing cell cycle arrest and induction of apoptosis by an intrinsic mechanism.