Hydroxyapatite (HA) and citric acid (Cit)-coordinated HA (Cit/HA) nanoparticle films with different nanospaces were used to examine the nanospacial effect on the protein adsorption behavior and initial osteoblast-like cell adhesion ability through the premise of the stability and ionic dissociation characteristics of the films in biological solution. In particular, the Cit/HA nanoparticle film with a nanospace of 4.2 nm could realize massive and stereoscopic adsorption of proteins due to its larger specific surface area and smaller nanospace as compared with the case of the HA nanoparticle film. It was also found that the α-helix and (β-sheet + β-turn) component ratios of the adsorbed fetal bovine serum proteins on the Cit/HA nanoparticle films increased as compared with the case of the HA nanoparticle film through the secondary structure analysis of the adsorbed proteins, which contributed to the good initial cell culture properties on the film surfaces. Therefore, we successfully realized the control of protein adsorption states using different nanospacial HA and Cit/HA nanoparticle films to achieve excellent initial cell culture properties, which would provide new insights into the creation of novel cell culture substrate surfaces in the regenerative medicine fields.