Cortactin binds F-actin and promotes cell migration. We showed earlier that cortactin is acetylated. Here, we identify SIRT1 (a class III histone deacetylase) as a cortactin deacetylase and p300 as a cortactin acetylase. We show that SIRT1 deacetylates cortactin in vivo and in vitro and that the SIRT1 inhibitor EX-527 increases amounts of acetylated cortactin in ovarian cancer cells. We also show that p300 acetylates cortactin in vivo and that cells lacking or depleted of p300 express lessacetylated cortactin than do control cells. Deletion analysis mapped the SIRT1-binding domain of cortactin to its repeat region, which also binds F-actin. Mouse embryo fibroblasts (MEFs) lacking sir2a (the mouse homolog of SIRT1) migrated more slowly than did wildtype cells. The expression of SIRT1 in sir2a-null cells restored migratory capacity, as did expression of a deacetylation-mimetic mutant of cortactin. SIRT1 and cortactin were more abundant in breast tumor tissue than in their normal counterparts, whereas SIRT1 expression inversely correlates with the ratio of acetylation cortactin versus total cortactin. These data suggest that deacetylation of cortactin is associated with high levels of SIRT1 and tumorigenesis. Finally, breast and ovarian cancer cell lines expressing an acetylation mimetic mutant of cortactin are less motile than that of control cells, whereas cells expressing the deacetylation mimetic mutant of cortactin migrate faster than that of control cells in Transwell migration assays. In summary, our results suggest that cortactin is a novel substrate for SIRT1 and p300 and, for the first time, a possible role for SIRT1 in cell motility through deacetylation of cortactin.