The aim of this study was to establish the role of serine/threonine protein phosphatase 2A (PP2A) in the survival of leukemic cells from patients with adult T cell leukemia (ATL), associated with human T cell leukemia virus type 1 (HTLV-1). In HTLV-1-infected T cell lines and ATL cells, okadaic acid (OkA), a potent PP2A inhibitor, induced decrease in cell viability and G1 cell cycle arrest by decreasing the expression levels of cyclin D2, cyclin-dependent kinase 4 and cyclin-dependent kinase 6, phosphorylation of pRb, and upregulation of p21, p27 and GADD45α. OkA-induced apoptosis was also due to the suppression of expression of Bcl-2, Bcl-xL and XIAP, and the activation of caspases-3, -8 and -9, and caspase-3 downstream mammalian STE20-like kinase 1 and H2AX. OkA inhibited nuclear factor-kappa B DNA binding and activated mitogen-activated protein (MAP) kinases. Other new PP2A-specific inhibitors, cytostatin and rubratoxin A, also induced decrease in cell viability through caspase-dependent mechanism. MAP kinase inhibitors confirmed the role of p38 MAP kinase in PP2A inhibitors-induced apoptosis. OkA resulted in the generation of reactive oxygen species, and exogenous antioxidant prevented activation of the indicated caspases. Finally, PP2A knockdown inhibited cell growth. The results showed that PP2A inhibition caused reactive oxygen species generation and affected distinct signaling pathways, resulting in the activation of H2AX and subsequent apoptotic cell death. These results suggest that PP2A is a potentially useful target in the treatment of ATL.