Abstract: Objective To investigate the effect of conditioned medium from rat RSC96 cells (RSC96-CM) on the proliferation of oligodendrocyte progenitor cells (OPCs) and explore the underlying mechanism. Methods OPCs isolated from the spinal cords of SD rats of embryonic day 15 using immunopanning were treated with RSC96-CM. The proliferation of OPCs was detected using 5-bromo-2’-deoxyuridine (BrdU) incorporation assay. The mRNA expressions of PDGF-AA and bFGF in RSC96 cells were detected using RT-PCR, and their protein concentrations in RSC96-CM were detected with enzyme-linked immunosorbent assay (ELISA). The effects of PDGF-AA and bFGF in RSC96-CM on OPC proliferation and the roles of ERK and JNK signaling pathways in RSC96-CM-induced OPC proliferation were determined by application of their specific inhibitors. Results The percentage of BrdU+ OPCs was significantly increased in response to treatment with RSC96-CM (P< 0.05), reaching the peak level when 50% RSC96-CM was added in the cell culture. RSC96 cells expressed a substantial amount of PDGF-AA and bFGF mRNAs, and PDGF-AA and bFGF protein concentrations in RSC96-CM were higher than those in a conditioned medium (B104CM) we used previously by 0.87 and 0.92 folds, respectively. Both the specific inhibitor of PDGFR signal pathway (AG1295) and the specific inhibitor of bFGFR signal pathway (PD173074) significantly attenuated RSC96-CM-induced OPC proliferation. The specific inhibitors of ERK signal pathway (U0126) and JNK signal pathway (SP600125) significantly decreased the percentage of BrdU + cells in RSC96-CM-induced OPCs (P<0.01). Conclusion RSC96-CM can effectively promote OPC proliferation, possibly as a result of PDGF-AA and bFGF secretion by RSC96 cells to activate ERK1/2 and JNK signaling pathways. RSC96- CM can be used as a routine stimulator for promoting OPC proliferation.