Gold (Au) is long considered as a biocompatible metal, and gold nanoparticles (AuNPs ~5 nm) were recently reported to scavenge free radicals. The effect of Au embedded in a polymeric material is less investigated compared with that of silver. In this study, nanocomposites from polyurethane (PU) and 43.5 or 174 ppm of AuNPs were prepared from a waterborne process. The response of endothelial cells (ECs) to the PU-Au nanocomposites was investigated in vitro and in vivo. ECs on PU-Au nanocomposites showed lamellipodia formation and better cell proliferation. The activation of proteins in ECs grown on PU-Au nanocomposites was analyzed by two-dimensional gel electrophoresis and confirmed by Western blot. The new protein identified through this procedure was valosin-containing protein (VCP), which is known to have immunomodulating effect. VCP was upregulated by PU-Au 43.5 ppm and PU-Au 174 ppm, but more in PU-Au 43.5 ppm. This suggested that the dispersion of AuNPs in the polymer matrix may be more important than the loading amount. PU-Au catheters implanted in rat blood vessels showed less foreign body reaction and more extensive EC coverage than the control PU catheters. The good in vivo biocompatibility of PU-Au may be associated with the anti-inflammatory effect of PU-Au. Based on this study, AuNPs may serve as an antioxidant additive for biomedical polymers.