Phostensin (PTS) encoded by KIAA1949 is a scaffold protein. It can bind to protein phosphatase 1 (PP1), F-actin, Eps 15 homology domain-containing protein 1 (EHD1) and EHD4. PTS contains both PP1-binding and actin-binding motifs. Most EHD-binding proteins contain a consensus motif, Asn-Pro-Phe (NPF) motif, which interacts with the C-terminal EH domain of EHD proteins. Nevertheless, the NPF motif is absent in PTS. The binding motif for PTS to associate with EHD1 (or EHD4) remains unknown. Here, we used structure-function, far western blotting and GST pull-down assays to investigate this issue and identified that PTS-α binds to EHD1 (or EHD4) through the region of residues 60-80 which contains a consensus motif, ⁶⁴ILV(X) ₄(L/V)RL ⁷⁴S. This novel motif was also found in the vacuolar protein sorting 35 (vps35) of retromer, an EHD1-associating complex and regulating retrograde trafficking, Replacement of ⁶⁴ILV(X) ₄(L/V)RL ⁷⁴S with ⁶⁴AAA(X) ₄(L/V)RL ⁷⁴S or with ⁶⁴ILV(X) ₄AEA ⁷⁴A significantly reduces the binding efficiency of PTS-α to either EHD1 or EHD4. Overexpression of the PTS-β in 293T cells attenuated the endocytic trafficking of transferrin. However, this attenuation of transferrin in endocytic trafficking was disrupted when 293T cells overexpressed the mutant PTS-β with a defective EHD-binding motif, suggesting that PTS-β can regulate the endocytic recycling via associating with EHD1 or EHD4.