It is widely held that growth factor signaling is terminated by lysosomal degradation of its activated receptor, and the endocytosed growth factor is transported to lysosomes. Nuclear targeting is another important pathway through which signals of growth factors are mediated. However, mechanisms underlying desensitization of nuclear targeting growth factors are poorly understood. Here we report that the nuclear targeting pathway is down-regulated by the proteasome system. Degradation of endocytosed midkine, a heparin-binding growth factor, was suppressed by both proteasome and lysosome inhibitors to similar extents. By contrast, a proteasome inhibitor, but not lysosome ones, accelerated the nuclear accumulation of midkine. An expression vector of signal sequence-less midkine, which is produced in the cytosol, was constructed, as endocytosed midkine may be translocated to the cytosol from cellular compartments before entering the nucleus. The cytosol-produced midkine underwent proteasomal degradation and accumulated in the nucleus as endocytosed midkine did. It was polyubiquitinated, and its nuclear accumulation was enhanced by a proteasome inhibitor. We further dissected the midkine molecule to investigate roles in degradation and trafficking. The N-terminal half domain of midkine was much more susceptible to proteasomal degradation, while the C-terminal half domain was sufficient for nuclear localization. Together, these data highlight the desensitization of nuclear targeting by growth factors, and indicate a critical role of the proteasome system in it.