Abstract: Objective To establish a simple and economic method for in vitro culture of tolerogenic dendritic cells (Tol-DC) from mouse bone marrow to facilitate further research of the application of Tol-DC in autoimmune disease. Methods The dendritic cells were derived from in vitro culture of the precursor cells from mouse bone marrow with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin (IL)-4, respectively. Eighteen hours before completion of cell culture, lipopolysaccharide (LPS) was added in the medium for stimulating rmGM-CSF-treated cells, but not IL-4-treated cells. The cells were collected on day 6, and the cell morphology, phenotype and immunofunction were analyzed. Results DC cultured in vitro had a CD11c+ expression rate of 76.67% with typical morphology. The DCs without LPS treatment were characterized by moderate expression of class II major histocompatibility complex (MHCⅡ) and low expression of costimulatory molecules, while those with LPS treatment had high expressions of MHCⅡand costimulatory molecules. Conclusion Immature tolerogenic DCs can be derived in large quantity using this method with the potential for use in the treatment of autoimmune diseases.