The establishment of a serum-free culture medium (SFM) for SSV-NIH3T3 cells is reported in this paper. In this medium DMEM/Ham's F12 is the fundamental element in basal medium, insulin and undefatty bovine serum albumin are key elements among the supplements. In this SFM SSV-NIH3T3 cells grow well, they keep the ability of secreting platelet-derived growth factor-like material into culture medium and causing tumor growth in nude mice.