Aim:The objective of this study was to investigate the effect of different vegetable oils on rumen fermentation and concentrations of beneficial cis-9 trans-11 C18:2 conjugated linoleic acid (CLA) and trans-11 C18:1 fatty acid (FA) in the rumen fluid in an in vitro condition.Materials and Methods:Six vegetable oils including sunflower, soybean, sesame, rice bran, groundnut, and mustard oils were used at three dose levels (0%, 3% and 4% of substrate dry matter [DM] basis) in three replicates for each treatment in a completely randomized design using 6 × 3 factorial arrangement. Rumen fluid for microbial culture was collected from four goats fed on a diet of concentrate mixture and berseem hay at a ratio of 60:40 on DM basis. The in vitro fermentation was performed in 100 ml conical flakes containing 50 ml of culture media and 0.5 g of substrates containing 0%, 3% and 4% vegetable oils.Results:Oils supplementation did not affect (p>0.05) in vitro DM digestibility, and concentrations of total volatile FAs and ammonia-N. Sunflower oil and soybean oil decreased (p<0.05) protozoal numbers with increasing levels of oils. Other oils had less pronounced effect (p>0.05) on protozoal numbers. Both trans-11 C18:1 FA and cis-9, trans-11 CLA concentrations were increased (p<0.05) by sunflower and soybean oil supplementation at 4% level with the highest concentration observed for sunflower oil. The addition of other oils did not significantly (p>0.05) increase the trans-11 C18:1 FA and cis-9, trans-11 CLA concentrations as compared to the control. The concentrations of stearic, oleic, linoleic, and linolenic acids were not altered (p>0.05) due to the addition of any vegetable oils.Conclusion:Supplementation of sunflower and soybean oils enhanced beneficial trans-11 C18:1 FA and cis-9, trans-11 CLA concentrations in rumen fluid, while sesame, rice bran, groundnut, and mustard oils were ineffective in this study.