Abstract: To investigate the effects of fatty acids on the proliferation and differentiation of preadipocytes from Nile tilapia (Oreochromis niloticus), 100 μmol/L Palmitic acid (PA), Oleic acid (OA), Linoleic acid (LA) and α-Linolenic acid (LNA) were used. SRB (Sulforhodamine B) staining and oil red O staining were used to detect cell proliferation and preadipocyte differentiation. The expression level of genes related to proliferation and differentiation was detected by Real-time qPCR. The results showed that exogenous fatty acids promoted the preadipocyte proliferation in 8 days, and significantly increased the expression level of proliferation related genes (c-fos and c-myc), lipolysis related genes (ATGL) and adipogenesis related genes (PPAR, CD36 and FAS) (P<0.05). In addition, fatty acid inhibited preadipocyte differentiation into adipocyte by decreasing lipid droplets area and increasing the lipid droplets number. During differentiation, expression level of β-oxidation related gene (CPT-1a) significantly increased (P<0.05), while the expression level of lipolysis related genes (ATG) and adipogenesis related genes (PPARγ and FAS) declined. Our results showed that exogenous fatty acid promoted the proliferation and inhibited preadipocyte differentiation. In the process of proliferation, excess fatty acids stored in the preadipocyte that could be metabolized by lipolysis and β-oxidation during adipogenesis to help cells adapt to high concentrations of fatty acids in the environment. While during the differentiation, the exogenous fatty acids can inhibit adipogenesis and lipolysis of adipocyte and promote β-oxidation to reduce adipocyte differentiation.