Dihydropyrimidinase-like 3 (DPYSL3) and GAP43 are both involved in neurite outgrowth, a crucial process for the differentiation of neurons. The present study shows for the first time that DPYSL3 co-localizes with GAP43 in primary cortical neurons. Further co-immunoprecipitation and overlay assay showed the ability of both recombinant and endogenous DPYSL3 to bind GAP43, indicating a specific interaction between DPYSL3 and GAP43 in primary cortical neurons.