A completely randomized block experiment was designed to investigate the effect of rubber seed oil on in vitro rumen fermentation, concentration of unsaturated fatty acids and gas production. Rubber seed oil was added to total mixed ration (TMR) at the rate of 0, 1, 2, 3 and 4% dry matter of the diet (4 replicates per treatment). The culture medium consisted of 25 mL rumen fluid collected from 3 fistulated Holstein cows, 50 mL basal medium and 0.5 g TMR (48% forage and 52% concentrate), and each treatment was incubated for 24 h at 39°C. Gas production, fermentation kinetics, in vitro dry matter digestibility (IVDMD), neutral detergent fiber degradation (DNDF), acid detergent fiber degradation (DADF) and volatile fatty acids (VFA) were determined. The results showed as follows, gas production, pH, NH₃-N, IVDMD, DNDF, DADF, total and individual VFAs were not affected by rubber seed oil (P > 0.05). However, the DNDF tended to decrease linearly with increasing levels of rubber seed oil (P < 0.1). After 24 h incubation, 3% and 4% DM of rubber seed oil increased the concentrations of t11C18:1, c9c12C18:2, c9t11CLA, C22:2 and total fatty acids (TFA) (P < 0.05), 4% DM of rubber seed oil increased the concentrations of t9C18:1, C18:3, t10c12CLA (P < 0.05). Rubber seed oil seemed to enhance the accumulation of unsaturated fatty acids in rumen but did not affect rumen fermentation.