Gene silencing using small interfering RNAs (siRNAs) is a selective and promising approach for treatment of numerous diseases. However, broad applications of siRNAs are compromised by their low stability in a biological environment and limited ability to penetrate cells. Nanodiamonds (NDs) coated with cationic polymers can enable cellular delivery of siRNAs. Recently, we developed a new type of ND coating based on a random copolymer consisting of (2-dimethylaminoethyl) methacrylate (DMAEMA) and N-(2-hydroxypropyl) methacrylamide (HPMA) monomers. These hybrid ND-polymer particles (Cop+-FND) provide near-infrared fluorescence, form stable complexes with siRNA in serum, show low toxicity, and effectively deliver siRNA into cells in vitro and in vivo. Here, we present data on the mechanism of cellular uptake and cell trafficking of Cop+-FND : siRNA complexes and their ability to selectively suppress mRNA levels, as well as their cytotoxicity, viability and colloidal stability. We identified clathrin-mediated endocytosis as the predominant entry mechanism for Cop+-FND : siRNA into U-2 OS human bone osteosarcoma cells, with a substantial fraction of Cop+-FND : siRNA following the lysosome pathway. Cop+-FND : siRNA potently inhibited the target GAPDH gene with negligible toxicity and sufficient colloidal stability. Based on our results, we suggest that Cop+-FND : siRNA can serve as a suitable in vivo delivery system for siRNA.