The aim of this study was to investigate the effects of replacing linseed oil (LO) by fish oil (FO) on fatty acid (FA) biohydrogenation. Experiment was conducted by a continuous in vitro incubation system using 12 fermenters. Each fermenter was added 40 ml rumen fluid, 160 ml buffer, 2 g mixed substrates, and 50 mg FO plus LO blends. The treatments were included basal diet supplemented (DM basis) with 25 g/kg LO (25LO), or LO was replaced with FO at 5 (20LO), 10 (15LO) and 15 g/kg (10LO). Substitution of FO for LO decreased mean amounts of C18:0 (P=0.057), c-9,c-12 C18:2 and C18:3n-3 (P(Pformation of ¬ruminal c-9,t-11 CLA concentration. To improve ruminal concentrations of C18:3n-3, C20:5n-3, C22:6n-3, ¬c-9,t-11 CLA and reduce C18:0 concentration, 10 g/kg FO replaced for LO would be an ideal choose.