A three-dimensional scaffold of type I collagen and hydroxyapatite enriched with polycaprolactone nanofibers (Coll/HA/PCL), autologous mesenchymal stem cells (MSCs) in osteogenic media, and thrombocyte-rich solution (TRS) was an optimal implant for bone regeneration in vivo in white rabbits. Nanofibers optimized the viscoelastic properties of the Coll/HA scaffold for bone regeneration. MSCs and TRS in the composite scaffold improved bone regeneration. Three types of Coll/HA/PCL scaffold were prepared: an MSC-enriched scaffold, a TRS-enriched scaffold, and a scaffold enriched with both MSCs and TRS. These scaffolds were implanted into femoral condyle defects 6 mm in diameter and 10-mm deep. Untreated defects were used as a control. Macroscopic and histological analyses of the regenerated tissue from all groups were performed 12 weeks after implantation. The highest volume and most uniform distribution of newly formed bone occurred in defects treated with scaffolds enriched with both MSCs and TRS compared with that in defects treated with scaffolds enriched by either component alone. The modulus of elasticity in compressive testing was significantly higher in the Coll/HA/PCL scaffold than those without nanofibers. The composite Coll scaffold functionalized with PCL nanofibers and enriched with MSCs and TRS appears to be a novel treatment for bone defects. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2014.