Endocytosis of activated EGF receptor (EGFR) to specific endocytic compartments is required to terminate EGF signaling. Trafficking of EGFR relies on microtubule tracks that transport the cargo vesicle to their intermediate and final destinations and can be modulated through posttranslational modification of tubulin including acetylation. Na,K-ATPase maintains intracellular sodium homeostasis, functions as a signaling scaffold and interacts with EGFR. Na,K-ATPase also binds to and is regulated by acetylated tubulin but whether there is a functional link between EGFR, Na,K-ATPase and tubulin acetylation is not known.EGF-induced sodium influx regulates EGFR trafficking through increased microtubule acetylation. Increased sodium influx induced either by sodium ionophores or Na,K-ATPase blockade mimicked the EGF-induced effects on EGFR trafficking through histone deacetylase (HDAC) 6 inactivation and accumulation of acetylated tubulin. In turn, blocking sodium influx reduced tubulin acetylation and EGF-induced EGFR turnover. Knockdown of HDAC6 reversed the effect of sodium influx indicating that HDAC6 is necessary to modulate sodium-dependent tubulin acetylation.These studies provide a novel regulatory mechanism to attenuate EGFR signaling in which EGF modulates EGFR trafficking through intracellular sodium-mediated HDAC6 inactivation and tubulin acetylation.