An in vitro study was conducted to investigate the effect of propionate precursor (malate or fumarate) on fermentation characteristics, production of conjugated linoleic acid (CLA) and methane (CH₄) by rumen microbes when incubated with linoleic acid (C18:2) as a CLA precursor. Total CH₄ production for 12h incubation, however, was greatly reduced (P＜0.0001) by all the supplements compared to that of control, and its production from M-LA or F-LA treatment was smaller than that from LA treatment. Supplementation of M-LA or F-LA also increased concentrations of cis9, trans11-CLA (P＜0.039 - P＜0.001) for all incubation times and trans10,cis 12-CLA at 1h (P＜0.013), 3h(P＜0.036) and 12h (P＜0.025) incubation times compared to LA supplementation.