The objectives of this experiment were to examine rumen fermentation and biohydrogenation altered by a methionine analog, branched-chain volatile fatty acids, or their combination in a condition of feeding high polyunsaturated fatty acids. An in vitro batch culture was conducted to determine the biohydrogenation of linoleic acid at 2, 4, 8, and 24 h and pH and VFA at 24 h. Dietary treatments included a typical diet (50:50 of forage to concentrate on a DM basis; CON), CON with 3.0% linoleic acid (DM basis; LA), LA with 0.1% of a methionine analog (HMTBa; Rhodiment, Adisseo Inc.), LA with isobutyrate, iso-valerate, and 2-methyl-butyrate (1 mmol/L of each; BCVFA), and a combination of HMTBa and BCVFA (COMBO). Data were analyzed using MIXED procedures of SAS with incubation as a random effect and treatment, time, and their interaction were fixed effects. At 24 h incubation, LA did not affect pH compared with CON. Compared with LA, BCVFA, and COMBO decreased (P < 0.01) pH. Total VFA tended to be lower for LA vs. CON but greater (P < 0.10) for HMTBa, BCVFA, and COMBO compared with LA. Linoleic acid supplementation (i.e., LA, HMTBa, BCVFA, and COMBO) decreased acetate and increased (P < 0.01) propionate concentration compared with CON. Compared with LA, there were minimal changes in VFA for HMTBa and BCVFA. Dry matter and NDF digestibility was lower (P < 0.01) for LA vs. CON without a difference among LA, HMTBa, BCVFA, and COMBO. Compared with LA, BCVFA had minimal effects on profile of long chain fatty acids. However, HMTBa and COMBO increased (P < 0.01) 18:1 t11 and 18:2 c9t11 at 4 h of incubation and tended to increase (P < 0.10) these intermediates at 8, 12, and 24 h compared with LA. However, 18:1 t10 and 18:2 t10c12 were not affected by HMTBa vs. LA. In conclusion, linoleic acid at 3% of DM depressed feed fermentation, which was alleviated by supplementation of BCVFA according to decreased pH and increased total VFA. Supplementation of a methionine analog altered microbial biohydrogenation pathways of linoleic acid.