Several studies have investigated that linoleic acid (LA) and eicosapentaenoic acid (EPA) affect cell proliferation and lipid catabolic gene expression in mammals. To determine if LA and EPA increase duck cell proliferation and lipid catabolic gene expression, the authors exposed duck primary hepatocyte cultures to LA or EPA. The results showed that both LA and EPA increased cell proliferation in a dose-dependent manner (100 μM). The effect on specific cell-cycle phases was also studied; LA and EPA (100 μM) deceased the proportion of cells in the G0/G1 phase from 83 to 80.8 and 80.3%, respectively, concomitant with an increase in the proportion of S-phase cells (11.5 and 10.5 vs. 8%, respectively). The expression of PPAR-α and PPAR-α target genes, such as acyl-CoA oxidase (ACOX), lipoprotein lipase (LPL), liver fatty acid-binding protein (L-FABP), was examined by quantitative real-time PCR. The results showed that the expression of the PPAR-α, ACOX, and LPL genes increased significantly following LA and EPA exposure, but that the expression of L-FABP remained unchanged. This study provides the first characterization of LA- and EPA-induced cell proliferation and PPAR-α and PPAR-α target gene transcriptional responses in duck primary hepatocyte cultures.