Semaphorins are a family of secreted and membrane‐associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post‐synaptic density protein SAP90/PSD‐95. Using the yeast two‐hybrid system and coprecipitation assays we were able to show an interaction between the extreme C‐terminus of Sema4F and the PDZ domains of SAP90/PSD‐95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD‐95 in COS cells leads to translocation of SAP90/PSD‐95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C‐terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post‐synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD‐95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.