Abstract: Objective To discuss isolation and culture methods of primary rat skeletal muscle satellite cells and observe its characterization of proliferation and differentiation in vitro culture. Methods Primary muscle satellite cells of relative high purity were obtained by improved two-steps enzymatic digestion combined with pre-plating techniques. Marker of muscle-derived cell, desmin, was identified by histocytochemistry. The characterization of cell proliferation was studied by the way of MTT assay experiment. Differentiation of muscle satellite cell under different culture conditions in vitro was also studied. Results The purity of harvested muscle satellite cell was more than 90 percent. When cultured in vitro, the latent phase of the cell is at the lst to 2nd days, platform phase is at the 5th to 6th day. It shows strong proliferative ability in vitro culture and could fuse with each other into myotube cells without special induction. Conclusions Muscle satellite cells by in vitro culture were enough to meet study of tissue engineering and cell transplantation.