Continuing from our previous study, we hypothesized that combining electrical stimulation (ES) and three-dimensional (3D) culture would be a useful strategy to obtain more bioactive factors in conditioned medium (CM) derived from human mesenchymal stem cells (hMSC). Our aim in this study was to investigate the bone-healing capacity of CM derived from hMSC after 4 days of culture on a collagen sponge-exposed (CM-ES) or unexposed (CM-control; CM-CON) to ES in comparison with that of hMSC implantation. A cytokine assay of both CMs revealed the presence of cytokines, growth factors, and trophic factors. In vitro evaluation of both CMs showed increased cell growth and alkaline phosphatase activity of the hMSC, with little difference between CMs. We investigated the bone-healing effect using two bone disease models: bone defect and inflammatory bone loss. The calvaria defect was implanted with whole CM or 3D-precultured hMSC unexposed to ES. Microcomputed tomography analysis after 4 weeks indicated a twofold greater bone volume in the CM-CON and CM-ES groups than in the hMSC and vehicle groups, though we found no difference between the CM groups. However, CM-ES enhanced the bone healing of interleukin-1-induced bone loss to a level comparable with hMSC, whereas CM-CON did not. These results show that 3D-cultured CM had a greater or similar capacity for bone healing as treatment using hMSC transplantation, and CM-ES was especially effective against inflammatory bone loss. Thus, 3D-cultured CM with or without ES presents an encouraging alternative to MSC-based bone healing. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 311-320, 2018.