The WW domain-containing oxidoreductase (WWOX) gene which encompasses the common human fragile site FRA16D has been proposed as a putative tumor suppressor gene, and loss of WWOX expression has been found in several types of solid cancer. As the role of WWOX in human leukemia has not yet been fully elucidated, the present study examined the expression of WWOX in patients with different types of leukemia as well as in leukemia-derived cell lines. Based on the data, WWOX mRNA (WWOX) and protein (Wwox) were significantly reduced or absent in the leukemia patients as well as in the cell lines. In addition, a recombinant expression vector, pGC-FU-WWOX, was constructed and transfected WWOX cDNA into Jurkat cells (acute T-lymphoblastic leukemia) and K562 cells (chronic myeloid leukemia in erythroid crisis) which all lack endogenous Wwox. In vitro experiments indicated that restoration of Wwox in Jurkat and K562 cells significantly suppressed proliferation and colony formation. Of note, apoptosis was also induced by Wwox restoration. Furthermore, we traced the mechanisms underlying this process and found that Wwox restoration could trigger the mitochondrial pathway in leukemia. Our data provide evidence that WWOX exerts a role as a tumor suppressor gene in leukemia, possibly by inhibiting proliferation and promoting apoptosis via the mitochondrial pathway.