We examined the effects of different forms of methionine on rumen fermentation and biohydrogenation in a condition of feeding polyunsaturated fatty acids. An in vitro batch culture was conducted with the following dietary treatments: a typical diet (50:50 of forage to concentrate on a DM basis; CON), CON with addition of 3.0% linoleic acid (DM basis; LA), LA with 0.1% of d/l-methionine (MET), 0.1% of a methionine analog (HMTBa; Rhodimet, Adisseo Inc.). The biohydrogenation of linoleic acid at 2, 4, 8, and 24 h and pH, ammonia, and volatile fatty acids (VFA) at 24 h of incubation were determined. Data were analyzed using the MIXED procedure of SAS where incubation was random effect and treatment, time, and their interaction were fixed effect. At 24-h incubation, pH and ammonia were not affected by treatments. Total VFA concentration was not affected by treatments. However, LA decreased acetate and butyrate and increased (P = 0.02) propionate as proportion of total VFA. Compared with LA, HMTBa increased acetate and decreased (P = 0.02) propionate, but no difference in VFA composition between LA and MET were found. Dry matter digestibility was decreased (P < 0.05) for LA vs. CON without a difference among LA, MET, and HMTBa. Digestibility of NDF was highest (P < 0.05) for CON followed by MET, HMTBa, and then LA. At 2, 4, and 24 h of incubation, no or minimal differences in concentrations of linoleic acid and biohydrogenation intermediates were observed among LA, MET, and HMTBa. However, at 8 h of incubation, MET and HMTBa tended to increase (P = 0.06) 18:1 t11 and increased (P = 0.01) 18:2 c9t11 compared with LA. However, 18:1 t10 and 18:2 t10c12 were not affected by MET and HMTBa. In conclusion, linoleic acid at 3% of substrate DM depressed feed digestibility and altered acetate and propionate production. However, MET and HMTBa alleviated the negative effect of LA on fiber digestibility and HMTBa produced more acetate and less propionate compared with LA, which did not occur for MET. Changes in the biohydrogenation of linoleic acid by MET and HMTBa were small but altered biohydrogenation pathways.