The study was conducted to evaluate the effects of different supplemental levels of monensin on the hydrogenation of linoleic acid (C18∶2) and the synthesis of trans11-oleinic acid (t11-C18∶1) in culture medium, which contained C18∶2 as the source of fatty acids, by a in vitro batch culture method. Single-factor experimental design was used in the experiment. Avicel was used as the substrate. Monensin was added by its mass concentration to the substrate and the levels were 0 (control group), 10 (group M1), 20 (group M2), 30 (group M3), and 40 mg/kg (group M4), respectively. Samples were collected after 2, 4, 8, 16 and 24 h of incubation, four replicates at each time point. Contents of stearic acid (C18∶0), t11-C18∶1 and cis9, cis12-linoleic acid (c9, c12-C18∶2) were determined.The results showed as follows: the content of C18∶0 in culture medium was reduced significantly (P<0.05), meanwhile, t11-C18∶1 and c9, c12-C18∶2 contents were significantly increased (P<0.05) by the supplemention of monensin in vitro; content of t11-C18∶1 was increased as the supplemental level of monensin increased when it was higher than 10 mg/kg. The results indicate that the supplemention of monensin in culture medium can increase the t11-C18∶1 content, and the synthsis of t11-C18∶1 was enhanced as the increasing of monensin level after the level is higher than 10 mg/kg.［Chinese Journal of Animal Nutrition，2011，23（2）：343-348］