The objectives of this study were to determine optimal functions of silage inoculants as probiotic agents for fermentative processes of rumen microbes, thereby focusing on efficient fermentative grass silage utilization and enhanced conjugated linoleic acid (CLA) production in fermentation fluid. The inoculant Lactobacillus plantarum added to the grass silage diet was effective in increasing CLA concentrations in fermentation fluid and decreasing biohydrogenation of C18 fatty acids in RUSITEC. However,the other two inoculants (i.e. Enterococcus faecium, Lactobacillus fermentum) had the opposite effect, suggesting that silages inoculated with different inoculants induced changes in the rumen fatty acids metabolism which might be related to differencesin the extent of rumen biohydrogenation of polyunsaturated fatty acids in RUSITEC.