The secretion of Wnt signaling proteins is dependent upon a transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network (TGN) and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, C. elegans and the mouse. The sorting signals for the internalisation and trafficking of Wls have not been defined. Here we demonstrate that Wls internalisation requires clathrin and dynamin I, components of the clathrin mediated endocytosis pathway. Moreover, we have identified a conserved YXXϕ endocytosis motif in the third intracellular loop of the multi-pass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of WlsAEGL was rescued by the insertion of a classical YXXϕ motif in the cytoplasmic tail. Significantly, a Drosophila WlsAEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signalling. These findings demonstrate that YXXϕ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signalling proteins.