Bacterial nanocellulose (BNC) is a promising material for small-caliber artificial blood vessels, although promoting its anticoagulant properties with more rapid endothelialization would improve long-term patency. Silk fibroin nanoparticles (SFNP) were introduced into the luminal wall surface of BNC conduits both with and without heparin (Hep) through pressurization followed by fixation. Hep was introduced in two ways: (1) embedded within SF nanoparticles to form SF-HepNPs for construction of the BNC-SF-HepNP conduit and (2) chemically grafted onto BNC and BNC-SFNP to form BNC-Hep and BNC-SFNP-Hep conduits. Fourier transform infrared spectroscopy confirmed the formation of SF-HepNPs, although they did not incorporate into the fibrillar network due to their large size. Hep was successfully grafted onto BNC and BNC-SFNP, verified by toluidine blue staining. The hemocompatibility and cytocompatibility of the five samples (BNC, BNC-SFNP, BNC-SF-HepNP, BNC-Hep, and BNC-SFNP-Hep conduits) were compared in vitro. The heparinized BNC-Hep and BNC-SFNP-Hep conduits improved the anticoagulant properties, and BNC-SFNP-Hep promoted human umbilical vein endothelial cell proliferation but also controlled excessive human arterial smooth muscle cell proliferation, assisting rapid endothelialization and improving lumen patency. No significant inflammatory reaction or material degradation was observed after subcutaneous implantation for 4 weeks. Autogenous tissues were observed around the conduits, and cells infiltrated into the edges of all samples, the BNC-SFNP conduit causing the deepest infiltration, providing an appropriate microenvironment for angiogenesis when used in small-caliber blood vessel applications. Few inflammatory cells were found around the BNC-Hep and BNC-SFNP-Hep conduits. Thus, the anticoagulant properties of the BNC-SFNP-Hep conduit and its stimulation of endothelialization suggest that it has great potential in clinical applications as a small-caliber artificial blood vessel.