Delphinidin 3-sambubioside (Dp3-Sam), a Hibiscus anthocyanin, was isolated from the dried calices of Hibiscus sabdariffa L. Dp3-Sam could induce a dose-dependent apoptosis in human leukemia cells (HL-60) as characterized by cell morphology, DNA fragmentation, activation of caspase-3, -8, and -9, and inactivation of poly(ADP)ribose polymerase (PARP). Molecular data showed that Dp3-Sam induced Bid truncation, mitochondrial membrane potential (DeltaPsi(m)) loss, and cytochrome c release from mitochondria to cytosol. Moreover, Dp3-Sam caused a time- and dose-dependent elevation of intracellular reactive oxygen species (ROS) level in HL-60 cells, and antioxidants such as N-acetyl-L-cysteine (NAC) and catalase could effectively block Dp3-Sam-induced ROS generation, caspase-3 activity, and DNA fragmentation. These data indicate that Dp3-Sam might induce apoptosis in HL-60 cells through a ROS-mediated mitochondrial dysfunction pathway. These findings enhance our understanding for anticancer function of Hibiscus anthocyanins in herbal medicine.