The effect of casticin was investigated by focusing on cell viability, apoptosis induction and cell cycle arrest in HL-60 cells. Casticin induced a dose- and time-dependent decrease in cell viability associated with apoptosis induction and G2/M cell cycle arrest. The addition of SB203580, an inhibitor for p38 mitogen-activated protein kinase (MAPK), but not SP600125 [c-Jun NH2-terminal protein kinase (JNK) inhibitor] and PD98059 [extracellular signal-regulated kinase (ERK) inhibitor], abrogated casticin-induced cell cycle arrest and apoptosis associated with the activation of caspases including caspase-8, -9 and -3. Endogenous p38 MAPK activation was observed in untreated cells based on the detection of the expression levels of phospho-p38 MAPK, whereas casticin did not affect the degree of p38 MAPK activation. Interestingly, the addition of SB203580 suppressed casticin-induced phosphorylation of histone H3, a downstream molecule of the p38 MAPK signaling pathway and known to be involved in chromosome condensation during mitosis. More importantly, casticin induced upregulation of intracellular ATP levels. Although casticin induced intracellular reactive oxygen species, antioxidants failed to block casticin-mediated cytotoxicity, indicating that casticin-induced cytotoxicity appears to be independent of reactive oxygen species generation. Based on the fact that SB203580 has been reported to compete with ATP for binding to the active form of p38 MAPK, and consequently blocks the p38 MAPK activity in activating downstream molecules, these results suggest that casticin induces cytotoxicity associated with apoptosis and cell cycle arrest in HL-60 cells through the p38 MAPK pathway, in which intracellular ATP levels and phosphorylation of histone H3 play critical roles.