Abstract: We investigated the effects of eicosapentaenoic acid (EPA) on proliferation, differentiation and gene expression of lipid metabolism in grass carp (Ctenopharyngodon idellus) preadipocytes. The developmental process of grass carp preadipocytes was observed at 0, 4, 6 and 14d after seeding. The proliferation of the preadipocytes was detected by methyl thiazolyl tetrazolium (MTT) assay method. The differentiation degree of cell was detected by oil red O staining after treated with 0-100 mol/L EPA. The cell was treated with 100 mol/L for two days after the cell con?uence, and then RNA was isolated. The expression of peroxisome proliferation activated receptors (PPARs), lipoprotein lipase (LPL) and peroxisome proliferatoractivated receptor gamma coactivator-1 (PGC-1) were detected by real-time PCR. The results showed that grass carp preadipocytes could reach confluence on seeding for six days in growth medium. The proliferation of preadipocytes was significantly promoted after EPA treatment with various levels for two days (P0.05), and the promotion effect disappeared at 3d. In comparison with the control group, intracellular lipid accumulation was significantly decreased at 1d following the addition of EPA (P0.05). At the same time, the mRNA level of LPL and PGC-1 were also significantly elevated after treated with 100 mol/L EPA for two days (P0.05), but there was no significant difference of the gene expression of PPARs observed between groups. It could be concluded that EPA enhanced the proliferation and inhibited the differentiation of grass carp preadipocytes. The suppression function of EPA on adipocytes differentiation and lipid accumulation might be related to its regulation on the expression of the lipid-metabolism-related genes, such as PGC-1 and LPL.