BACKGROUND Previous methods for Kupffer cells (KCs) isolation require sophisticated skills and tedious procedures. Few studies have attempted to explore the self-renewal capacity of KCs in vitro. Therefore, the aim of this study was to establish a simple method for rat KCs isolation and further investigate the mitotic potential of KCs in vitro. METHODS KCs were obtained by performing one-step perfusion, enzymatic tissue treatment, differential centrifugation and selective adherence. The proliferation ability of cultured KCs was determined by MTT assay and Propidium Iodide FACS analysis. Phagocytic assay and ED-1, ED-2 immunofluorescence were used to identify cell phenotype. After stimulation with LPS, the expression of surface antigens (MHCII, CD40, CD80, and CD86) and the production of cytokines (NF-κB, TNF-α, IL-6 and IL-10) were measured for cell function identification. RESULTS KCs were isolated with certain numbers and reasonable purities. The KCs were able to survive until at least passage 5 (P5), and at P3 showed equally strong phagocytic activity as primary KCs (P0). After stimulation with LPS, the change in the expression of surface antigens and the production of cytokines for P3 cells was similar to that for P0 cells. CONCLUSIONS Our study provides a simple and efficient method for KCs isolation, and reveals that self-renewing KCs have the same phagocytic activity and functions as primary KCs.