Bcl-x(L), an anti-apoptotic Bcl-2 family member protein, contributes to the resistance against chemotherapies such as tubulin-binder treatment in many human tumors. Although Bcl-x(L) is phosphorylated after tubulin-binder treatment, the role of the phosphorylation and its responsible kinase(s) are poorly understood. Here, we identified Plk1 (polo-like kinase 1) as a Bcl-x(L) kinase. Same location of Bcl-x(L) and Plk1 was revealed by immunocytochemical analyses at M-phase in situ. Plk1 phosphorylates Bcl-x(L) in vitro, and we identified Plk1 phosphorylation sites in Bcl-x(L). When all of these phosphorylation sites were substituted to alanines, the anti-apoptotic activity of the Bcl-x(L) mutant against the apoptosis induced by pironetin, but not against ultraviolet-induced apoptosis, was increased. These observations suggest that Plk1 is a regulator of Bcl-x(L) phosphorylation and controls the anti-apoptotic activity of Bcl-x(L) during pironetin-induced apoptosis.