Nanocomposites based on superhydrophilic vertically aligned multi-walled carbon nanotubes (VAMWCNT-O 2 ) and nanohydroxyapatite (nHAp) are of great interest in bone regenerative medicine. The biomineralization using simulated body fluid (SBF) has been extensively studied to evaluate the bioactivity of biomaterials. Thus, the combination of nHAp and VAMWCNT-O 2 is attractive and promising. The aim of this study was to evaluate the in vitro calcification of nHAp/VAMWCNT-O 2 nanocomposites before and after the period of biomineralization in SBF. In vitro calcification of the extracellular matrix (ECM) of HOB cells in culture after 24 hours was investigated through the assay of alkaline phosphatase. These promising in vitro results validate biomineralized nHAp/VAMWCNT-O 2 as possible scaffolds for bone tissue regeneration.