Burn wound treatment is difficult and one of the most challenging problems in the clinic. Researchers have examined the applications of mesenchymal stem cells as a cell-based therapy for skin regeneration. But the role of human bone marrow mesenchymal stem cell conditioned medium (hBM-MSC-CM) in the treatment of burn injury remains unclear. This research aims at detecting whether hBM-MSC-CM can increase the wound healing of deep second-degree burns in male rats. In this study, 32 adult male rats per each time point were randomly divided into four groups: (1) control group, (2) sham group (DMEM), (3) common treatment group (CT), and (4) conditioned media group (CM). A 3 × 3 cm circular burn was created on the back of the rats. On postsurgical days 7, 15, and 28, the wound closure area of each wound was measured and then the skin samples were removed and analyzed using stereological methods. Wound closure area was significantly increased in the CM and CT groups on the 15th and the 28th day after burn injury compared to the control and DMEM groups. The stereological parameters and immunohistochemistry analysis of the wounds revealed significantly improved healing in the CM group compared to the control and other groups. It is concluded that these findings indicate that hBM-MSC-CM promotes skin wound healing by increasing cell proliferation, regulating collagen synthesis and collagen composition, and inducing angiogenesis at the injury site.