Tumor progression locus-2 (Tpl-2) kinase is a member of the mitogen-activated protein kinase kinase kinase family that has been implicated in cellular transformation. The enhanced expression of this protein has been shown to activate both the mitogen-activated protein kinase and c-Jun N-terminal kinase pathways. However, the molecular mechanisms responsible for the oncogenic potential of Tpl-2 are still largely unknown. Here, we showed that Tpl-2 interacted with p53 both in vitro and ex vivo. The overexpression of Tpl-2 inhibited the epidermal growth factor (EGF)-induced p53 phosphorylation (Ser15) through upregulating the activity of protein phosphatase 2A, which interacted with p53 stimulated by EGF. Also, the EGF-induced p53 activity was suppressed in the Tpl-2 wild-type (WT)-transfected HEK 293 cells, but had no effect in the Tpl-2-mutant (S413A)-transfected cells. Furthermore, introduction of small interfering RNA-Tpl-2 into HEK 293 cells resulted in decreased cell viability compared with only adenovirus-p53-infected cells. In addition, the Tpl-2 WT, but not Tpl-2 mutant (S413A), showed increased EGF-induced c-fos promoter activity, followed by activator protein 1 (AP-1) transactivation activity, which was associated with the cell transformation prompted by the H-Ras-Tpl-2-AP-1 signaling axis. These results indicated that the Ser413 of Tpl-2 plays an important role in EGF-induced carcinogenesis as well as inactivation of the p53.