Cisplatin is an effective DNA-damaging antitumor agent employed for the treatment of various human cancers. In this study, we report that Cisplatin activates PKB/Akt in several cancer cell lines and that this activation is mediated by EGFR, Src and PI3-kinase. Inhibition of PI3-kinase activity decreases the survival of the cells exposed to Cisplatin, suggesting that Cisplatin-induced PKB/Akt activation may lead to Cisplatin resistance. While investigating the EGFR-dependent PKB/Akt activation in MDA-MB-468 cells, we found that the EGFR receptor undergoes a gel mobility shift upon Cisplatin treatment, which is mediated by p38(MAPK). An EGFR, in which threonine 669 was mutated to alanine (A669), is phosphorylated by p38(MAPK) to a much lesser extent, suggesting that threonine 669 is a p38 phosphorylation site. We found that Cisplatin induces EGFR internalization, which is mediated by p38(MAPK-)dependent phosphorylation of the receptor on threonine 669. Our results identify the EGFR as a new substrate of p38 and identify threonine 669 as a new phosphorylation site that regulates EGFR internalization. Together, these results suggest that Cisplatin has side effects, which may alter the signaling pattern of cancer cells and modulate the desired effects of Cisplatin treatment.