Objective To perform primary culture of glomerular mesangial cells, identify them from a mouse model of spontaneous lupus nephritis model and explore the optimal conditions for these procedures. Methods Nine control ICR mice and nine model MRL/lpr mice with spontaneous lupus nephritis were tested for autoantibodies using ELISA and urine protein was detected by bicinchoninic acid (BCA) assay at 12, 14 and 18 weeks of age. HE and Masson staining were used to observe the pathological changes of mice with lupus nephritis. Glomeruli were isolated using a cell sieve, while primary culture of glomerular mesangial cells was performed using the eugenic selection method. Glomerular mesangial cells of the MRL/lpr mice were identified based on cell morphology and immunocytochemical staining of α-smooth muscle actin and nephrin. Results In the model group, levels of anti-nuclear antibody and anti-double-stranded DNA antibodies significantly increased. Urine protein levels also increased significantly at 14 and 18 weeks of age. HE and masson staining of kidney tissue revealed pathological changes associated with lupus nephritis in the MRL/lpr mice from 12 weeks of age. Twenty-five days after seeding of the glomerular mesangial cells, those from the MRL/lpr mice gradually covered the bottom of the culture flasks, appearing star-shaped and fusiform by phase contrast microscopy. Immunocytochemistry showed that these cells were α-smooth muscle actin-positive and nephrin-negative, thus ruling out other glomerular intrinsic cells. Conclusion The primary culture of glomerular mesangial cells from mice with spontaneous lupus nephritis is developed and characterized.