Polo-like kinase 1 (Plk1), a serine/threonine protein kinase normally expressed in mitosis, is frequently up-regulated in multiple types of human tumors regardless of the cell cycle stage. However, the causal relationship between Plk1 up-regulation and tumorigenesis is incompletely investigated. To this end, using a conditional expression system, here we generated Plk1 transgenic mouse lines to examine the role of Plk1 in tumorigenesis. Plk1 overexpression in mouse embryonic fibroblasts prepared from the transgenic mice led to aberrant mitosis followed by aneuploidy and apoptosis. Surprisingly, Plk1 overexpression had no apparent phenotypes in the mice. Given that no malignant tumor formation was observed even after a long period of Plk1 overexpression, we reasoned that additional factors are required for tumorigenesis in Plk1-overexpressing mice. Because Plk1 can directly participate in the regulation of the DNA damage response (DDR) pathway, we challenged Plk1-overexpressing mice with ionizing radiation (IR) and found that Plk1-overexpressing mice are much more sensitive to IR than their wild-type littermates. Analysis of tumor development in the Plk1-overexpressing mice indicated a marked decrease in the time required for tumor emergence after IR. At the molecular level, Plk1 overexpression led to reduced phosphorylation of the serine/threonine kinases ATM and Chk2 and of histone H2AX after IR treatment both in vivo and in vitro Furthermore, RNA-Seq analysis suggested that Plk1 elevation decreases the expression of several DDR genes. We conclude that Plk1 overexpression may contribute to tumor formation by both inducing chromosomal instability and suppressing the DDR pathway.