An association between in vitro and in vivo studies has been demonstrated for the first time, using a novel nanohydroxyapatite/superhydrophilic vertically aligned multiwalled carbon nanotube (nHAp/VAMWCNT-O2) nanocomposites. Human osteoblast cell culture and bone defects were used to evaluate the in vitro extracellular matrix (ECM) calcification process and bone regeneration, respectively. The in vitro ECM calcification process of nHAp/VAMWCNT-O2 nanocomposites were investigated using alkaline phosphatase assay. The in vivo biomineralization studies were carried out on bone defects of C57BL/6/JUnib mice. Scanning electron microscopy, micro-energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and X-ray difractometry analyses confirmed the presence of the nHAp crystals. nHAp/VAMWCNT-O2 nanocomposites induced in vitro calcification of the ECM of human osteoblast cells in culture after only 24 h. Bone regeneration with lamellar bone formation after 9 weeks was found in the in vivo studies. Our findings make these new nanocomposites very attractive for application in bone tissue regeneration.