We investigated the dose-dependent biphasic effects of arsenic disulfide (As₂S₂) on the differentiation and apoptosis of HL-60 cells. Cellular reactive oxygen species, glutathione, p38 mitogen-activated protein kinase (MAPK), as well as cell differentiation and apoptosis in HL-60 cells were assessed by flow cytometric analysis. The mean percentage of CD11b-positive cells induced by 8 μM As₂S₂ was more than 3 times higher than that induced by 16 μM As₂S₂. Eight μM As₂S₂ did not induce cell apoptosis, while 16 μM As₂S₂ induced apoptosis and decreased cell viability. Both 8 and 16 μM As₂S₂ increased cellular reactive oxygen species. Treatment with 16 μM As₂S₂ decreased the cellular glutathione levels at 1 h and 3 h after the exposure. The mitochondrial membrane potential depletions were observed in both 8 μM As₂S₂-induced differentiation and 16 μM As₂S₂-induced apoptosis. p38 MAPK inhibition enhanced As₂S₂-induced differentiation, but had little influence on As₂S₂-induced apoptosis. A moderate oxidative stress induced by 8 μM As₂S₂ can promote As₂S₂-induced differentiation, whereas more severe oxidative stress caused by glutathione depletion by 16 μM As₂S₂ reduced mitochondrial membrane potential, resulting in differentiation attenuation and apoptosis enhancement. p38 MAPK activation resulting from oxidative stress seems to be implicated in negative regulation of cell differentiation, rather than apoptosis, in As₂S₂-treated HL-60 cells.