Abstract: The objective of this study was to establish the optimal conditions for enrichment and isolating of ruminal trans11 18:1 (TVA) hydrogenating bacteria in vitro. TVA was added into anaerobic mediums with different final concentrations (0, 30, 40, 50, 60 μg·mL-1). Ruminal microbes mixed were inoculated into mediums for continuous cultivation, samples were collected every 4 h and used for detecting TVA concentration and OD values. In addition, TVA was added into the anaerobic medium with a final concentration of 50 μg·mL-1, then the cultures were transfered for six generations for enrichment. Changes of the bacterial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling. Different DGGE bands of the enrichment cultures were identified by 16S rDNA gene sequencing. The result showed that the concentration of TVA in mediums was significantly decreased at 4 h during continuous incubation and then maintained constancy at 12 h. After incubation for 12 h, degradation rates of TVA with the concentration for 50 and 60 mg·mL-1 were higher than that of other concentration. Besides, the OD value of the culture medium increased and then decreased, and reached the peak at 12 h and then decreased. The amounts and types of suspected TVA hydrogenated strains significantly increased at the 4th generation. Sequencing results for the bands showed that most of them belong to Lactobacillus gasseri and uncultured bacterium. This study determined the suitable TVA adding amount, transfer time and generations for enrichment culture of ruminal TVA hydrogenating bacteria, and laid foundation for the subsequent selective separating culture of TVA hydrogenating bacteria.