Ligand-induced receptor down-regulation by endocytosis is a critical process regulating the intensity and duration of receptor tyrosine kinase signaling. Ubiquitylation of specific receptor tyrosine kinases, for example, the epidermal growth factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl, provides a sorting signal for lysosomal degradation and leads to termination of receptor signaling. Cortactin, which couples the endocytic machinery to dynamic actin networks, is encoded by EMS1, a gene commonly amplified in breast and head and neck cancers. One mechanism whereby cortactin overexpression contributes to tumor progression is by enhancing tumor cell invasion and metastasis. However, in this study, we show that overexpression of cortactin in HeLa cells markedly inhibits ligand-induced down-regulation of the EGFR. This is independent of alterations in receptor autophosphorylation and correlates with impaired c-Cbl phosphorylation and association with the EGFR, reduced EGFR ubiquitylation, and sustained EGF-induced extracellular signal-regulated kinase activation. Furthermore, analysis of a panel of head and neck squamous cell carcinoma (HNSCC) cell lines revealed that cortactin overexpression is associated with attenuated ligand-induced EGFR down-regulation. Importantly, RNAi-mediated reduction of cortactin expression in an 11q13-amplified HNSCC cell line accelerates EGFR degradation. This represents the first demonstration of modulation of growth factor receptor signaling by cortactin. Moreover, enhanced EGFR signaling due to cortactin overexpression may provide an alternative explanation for EMS1 gene amplification in human cancers.