Mesenchymal stem cells (MSCs) are an attractive cell source for tissue regeneration and repair. However, their low differentiation efficacy currently impedes the development of MSC therapy. Therefore, in this study, we investigated the effects of differentiation-inducing factor-1 (DIF-1) on the differentiation efficacy of bone marrow-derived MSCs (BM-MSCs) into adipogenic or osteogenic lineages. BM-MSCs, which were obtained from Sprague-Dawley rats, were positive for the MSC markers (CD29, CD73, and CD90). DIF-1 alone neither affected cell surface antigen expression nor induced adipogenic or osteogenic differentiation. However, DIF-1 significantly enhanced the effects of adipogenic differentiation stimuli, which were evaluated as the number of oil red-O positive cells and the expression of adipocyte differentiation markers (peroxisome proliferator-activated receptor gamma, adipocyte fatty acid-binding protein, and adiponectin). In contrast, DIF-1 significantly attenuated the effects of osteogenic differentiation stimuli, which were evaluated as alizarin red-S positive calcium deposition, and the expression of osteoblast differentiation markers alkaline phosphatase, runt-related transcription factor 2, and osteopontin. We further investigated the mechanism by which DIF-1 affects MSC differentiation efficacy and found that glycogen synthase kinase-3 was the main factor mediating the action of DIF-1 on the adipogenic differentiation of BM-MSCs, whereas it was only partially involved in osteogenic differentiation. These results suggest that DIF-1 supports MSC differentiation toward the desired cell fate by enhancing the differentiation efficacy.