Stem cell transplantation is a promising therapeutic strategy that includes both cell therapy and tissue engineering for the treatment of many regenerative diseases; however, the efficacy and safety of stem cell therapy depend on the cell type used in therapeutic and translational applications. In this study, we validated the hypothesis that human nasal turbinate-derived mesenchymal stem cells (hTMSCs) are a potential therapeutic source of adult stem cells for clinical use in bone tissue engineering using three-dimensional (3D) cell-printing technology. hTMSCs were cultured and evaluated for clinical use according to their cell growth, cell size, and preclinical safety and were then incorporated into a multicompositional 3D bioprinting system and investigated for bone tissue regeneration in vitro and in vivo. Finally, hTMSCs were compared with human bone marrow-derived MSCs (hBMSCs), which are the most common stem cell type used in regenerative medicine. hTMSCs from three different donors showed greater and faster cell growth than hBMSCs from two different donors when cultured. The hTMSCs were smaller in size than the hBMSCs. Furthermore, the hTMSCs did not exhibit safety issues in immunodeficient mice. hTMSCs in 3D-printed constructs (3D-hTMSC) showed much greater viability, growth, and osteogenic differentiation potential in vitro than hBMSCs in 3D-printed constructs (3D-hBMSC). Likewise, 3D-hTMSC showed better cell survival and alkaline phosphatase activity and greater osteogenic protein expression than 3D-hBMSC upon subcutaneous implantation into the dorsal region of nude mice. Notably, in an orthotopic model involving implantation into a tibial defect in rats, implantation of 3D-hTMSC led to greater bone matrix formation and enhanced bone healing to a greater degree than implantation of 3D-hBMSC. The clinically reliable evidence provided by these results is underlined by the potential for rapid tissue regeneration and ambulation in bone fracture patients implanted with 3D-hTMSC.