Flotillin-1 and flotillin-2 co-assemble into plasma membrane microdomains that are involved in the endocytosis of molecules such as glycosyl phosphatidylinositol (GPI)-linked proteins. Previous studies suggest that budding of flotillin microdomains from the plasma membrane is a tightly regulated process. Here, we demonstrate that endocytosis of flotillins is regulated by the Src family kinase Fyn. The Src kinase inhibitor PP2 prevents EGF-induced flotillin internalisation, and EGF-induced internalisation does not occur in SYF cells lacking Src, Yes and Fyn. Expression of Fyn, but not Src or Yes, restores EGF-induced internalisation in SYF cells. Expression of an active form of Fyn but not other Src kinases is sufficient to induce redistribution of flotillins from the plasma membrane to late endosomes and lysosomes. Using two partial Fyn constructs that form a functional kinase upon addition of rapamycin to cells, we show that flotillin internalisation from the plasma membrane occurs shortly after Fyn activation. Tyr160 in flotillin-1 and Tyr163 in flotillin-2 are directly phosphorylated by Fyn, and mutation of these residues to phenylalanine prevents Fyn-induced flotillin internalisation. Uptake of the GPI-linked protein CD59 is reduced by expression of the phenylalanine-mutated flotillins. These data establish uptake of flotillin microdomains as a tyrosine-kinase-regulated endocytic process.