Hydroxyapatite (HA) layers are appropriate biomaterials for use in the modification of the surface of implants produced inter alia from a Ti6Al4V alloy. The issue that must be solved is to provide implants with appropriate biointegration properties, enabling the permanent link between them and bone tissues, which is not so easy with the HA layer. Our proposition is the use of the intermediate layer ((IL) = TiO2, and titanate layers) to successfully link the HA coating to a metal substrate (Ti6Al4V). The morphology, structure, and chemical composition of Ti6Al4V/IL/HA systems were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy-dispersive X-ray spectrometry (EDS). We evaluated the apatite-forming ability on the surface of the layer in simulated body fluid. We investigated the effects of the obtained systems on the viability and growth of human MG-63 osteoblast-like cells, mouse L929 fibroblasts, and adipose-derived human mesenchymal stem cells (ADSCs) in vitro, as well as on their osteogenic properties. Based on the obtained results, we can conclude that both investigated systems reflect the physiological environment of bone tissue and create a biocompatible surface supporting cell growth. However, the nanoporous TiO2 intermediate layer with osteogenesis-supportive activity seems most promising for the practical application of Ti6Al4V/TiO2/HA as a system of bone tissue regeneration.