The effect of rumen fluid (RF) inoculum either unadapted (U-RF) or adapted (A-RF) to Saccharomyces cerevisiae fermentation product (SCFP; Diamond V Original XPC) on the biohydrogenation (BH) of unsaturated fatty acids (FA) at 2 pH levels and starch sources with different fermentabilities (SF) were evaluated. Rumen inocula for batch cultures were collected at the end of each 4-wk feeding period in a crossover design (n = 6). U-RF or A-RF cultures (4 replicates/treatment) were incubated for 24 h at pH 5.8 or 6.2 and included alfalfa hay (55% of DM), and either dry ground corn (DC) or high moisture corn (HMC) as starch sources (45% of DM). The alfalfa hay was treated with corn oil (2% of DM) to increase the total unsaturated FA content of cultures. Effects of RF, culture pH, SF, and their interactions were determined. For main effects of treatments, A-RF compared with U-RF and pH 6.2 compared with pH 5.8 increased extent of BH for cis-9,cis-12 18:2 (41 vs. 38% and 47 vs. 32%, respectively; both P < 0.001) and NDFD (14 vs. 12% and 16 vs. 10%, respectively; both P < 0.001). Compared with DC, HMC increased BH extent for cis-9,cis-12 18:2 (41 vs. 39%, P = 0.06), and decreased NDFD (12 vs. 14%, P < 0.001). Overall, compared with U-RF, A-RF decreased trans-10,cis-12 18:2 (CLA) by 17% (P < 0.001), and increased trans-10 18:1 by 10% (P < 0.001). HMC compared with DC, and pH 5.8 compared with pH 6.2, increased content of CLA by 15 and 56%, respectively (both P < 0.01), and increased trans-10 18:1 by 23 and 26%, respectively (both P < 0.001). RF interacted with SF for CLA and trans-10 18:1 (both interactions P < 0.01); in particular, for cultures containing HMC, A-RF decreased the content of CLA and increased the content of trans-10 18:1 compared with U-RF. Results demonstrate that under the conditions tested, rumen fluid from cows adapted to SCFP (A-RF) had positive effects on the extent of BH of unsaturated FA and NDFD. A-RF decreased CLA across culture pH, especially when HMC was the starch source.