The aim of this work was to assess the effect of different stabilizing agents, for example, polyethylenimine (PEI) and polyvinylpyrrolidone (PVP), on gold nanoparticles (AuNPs) and their influence on equine platelet activation and release of particular growth factors. The gold nanoparticles were produced by chemical reduction of chloroauric acid. UV-Vis spectroscopy confirmed the presence of gold nanoparticles in investigated solutions. The AuNPs were incubated with whole blood at various concentrations. The morphology of platelets in PRP prepared from the blood incubated with AuNPs was characterized by scanning transmission electron microscopy, whereas the concentrations of growth factors and cytokines were evaluated by ELISA assays. The most promising results were obtained with equine platelets incubated with 5% AuNPs stabilized by PEI, which lead to secretion of bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor (VEGF), and fibroblast growth factor 1 (FGF-1) and simultaneously cause decrease in concentration of interleukin-1 alpha (IL-1α). The qRT-PCR confirmed ELISA test results. The incubation with 5% AuNPs stabilized by PEI leads to upregulation of BMP-2 and VEGF transcripts of mRNA level and to downregulating expression of interleukin-6 (IL-6). Obtained data shed a promising light on gold nanoparticle application for future regenerative medicine application.