Background Given the limitations of current therapies for the reconstruction of bone defects, regenerative medicine has arisen as a new therapeutic strategy along with mesenchymal stem cells (MSCs), which, because of their osteogenic potential and immunomodulatory properties, have emerged as a promising alternative for the treatment of bone injuries. In vivo studies have demonstrated that MSCs have a positive effect on regeneration due to their secretion of cytokines and growth factors that, when collected in conditioned medium (MSC-CM) and applied to an injured tissue, can modulate and promote the formation of new tissue. Objective To evaluate the effectiveness of application of conditioned medium derived from mesenchymal stem cells in bone regeneration in animal and human models. Methods We conducted a systematic review with a comprehensive search through February of 2018 using several electronic databases (MEDLINE, EMBASE, SCOPUS, CENTRAL (Ovid), and LILACS), and we also used the “snowballing technique”. Articles that met the inclusion criteria were selected through abstract review and subsequent assessment of the full text. We assessed the risk of bias with the SYRCLE and Cochrane tools, and three meta-analyses were performed. Results We included 21 articles, 19 of which used animal models and 2 of which used human models. In animal models, the application of MSC-CM significantly increased the regeneration of bone defects in comparison with control groups. Human studies reported early mineralization in regenerated bones, and no bone resorption, inflammation, nor local or systemic alterations were observed in any case. The meta-analysis showed an overall favorable effect of the application of MSC-CM. Conclusions The application of MSC-CM to bone defects has a positive and favorable effect on the repair and regeneration of bone tissue, particularly in animal models. It is necessary to perform additional studies to support the application of MSC-CM in clinical practice.