A novel porous beta-tricalcium phosphate /collagen fibers (β-TCP/CF) composite, having a well-dispersed nano-sized β-TCP in collagen matrix, was developed by a wet-chemical method. The nano-composite was compared to conventional β-TCP on cytocompatibility by cell attachment, proliferation, alkaline phosphotatse (AKP) activity and scanning electron microscopy (SEM) analysis. These in vitro assays showed that the β-TCP/CF composite elicited cell adhesion and proliferation better then controls. Moreover experiments on osteoblast-like cells showed improved cell growth with the highly characterized nanophase structure. SEM micrographs also showed that the nano-sized composite exhibited much more viable cells in attachment on the surface compared with the controls. At 1, 3 and 5 days, AKP activity was not significant different for the tested and control samples, while at 7 day after culture, significantly increased AKP activity was observed for β-TCP/CF than for the control. The in vitro results obtained confirmed the remarkable improvement of cell adhesion and proliferation of the nano-sized β-TCP/CF composite, which may be a new promising candidate for tissue engineered bone substitute.