Rapid liver regeneration is required after living-donor liver transplantation and oncologic liver resections to warrant sufficient liver function and prevent small-for-size syndrome. Recent evidence highlights the therapeutic potential of mesenchymal stem cells (MSC) for treatment of toxic liver injury, but whether MSC and their secreted factors stimulate liver regeneration after surgical injury remains unknown. Therefore, the aim of this study is to investigate the effect of human liver-derived MSC-secreted factors in an experimental liver resection model. C57BL/6 mice were subjected to a 70% partial hepatectomy and treated with either concentrated MSC-conditioned culture medium (MSC-CM) or vehicle control. Animals were analyzed for liver and body weight, hepatocyte proliferation, and hepatic gene expression. Effects of MSC-CM on gene expression in a human hepatocyte-like cell line (Huh7 cells) were analyzed using genome-wide gene expression arrays. Liver regeneration was significantly stimulated by MSC-CM as shown by an increase in liver to body weight ratio and hepatocyte proliferation. MSC-CM upregulated hepatic gene expression of cytokines and growth factors relevant for cell proliferation, angiogenesis, and anti-inflammatory responses. In vitro, treatment of Huh7 cells with MSC-CM significantly altered expression levels of ~3,000 genes. Functional analysis revealed strong effects on networks associated with protein synthesis, cell survival, and cell proliferation. This study shows that treatment with MSC-derived factors can promote hepatocyte proliferation and regenerative responses in the early phase after surgical resection. MSC-CM may represent a feasible new strategy to promote liver regeneration in patients undergoing extensive liver resection or after transplantation of small liver grafts.