Abstract Corn supplementation can enhance the function of rumen and mitigate methane production. Thus, this study aimed to evaluate in vitro rumen digestibility, fermentation parameters and fatty acid biohydrogenation of palm kernel cake-based (PKC) diet substituted with different levels of corn. Corn was substitution into PKC basal diet at the levels; T1= (0% corn + 75.3% PKC), T2= (5% corn + 70.3% PKC) and T3= (10% corn + 65.3% PKC) of the diet. Rumen liquor was obtained from four fistulated Dorper sheep and incubated with 200 mg of each treatment for 24hrs and 72hrs. Net gas production, fermentation kinetics, in vitro organic matter digestibility (IVOMD), in vitro dry matter digestibility (IVDMD), volatile fatty acids (VFA), rumen microbial population and fatty acid biohydrogenation were determined. The results of the in vitro study showed that production of gas increased from 0 hr until 9 hrs with T2 having the highest gas production during this phase. After 48 hrs, the gas production began to decrease gradually with increase in incubation time. No significant differences were observed in the IVDMD, IVOMD, NH3-N, pH and VFA at 72 hrs. However, higher significant methane gas (CH4) production was observed in T3 when compared with T1 and T2. Microbial population did not differ significantly between treatment groups for total bacteria, F. succinogenes and R. flavefaciens. The rates of biohydrogenation were not affected by corn substitution although a significant difference was observed in that of C18:1n9. In conclusion, corn substitution maintained fermentation characteristics with increasing of unsaturated fatty acids.