The objective of this study was to investigate changes in rumen environment when animals are fed a diet containing 2-hydroxy-4-(methylthio) butanoate (HMTBa). Six cannulated Holstein steers were utilized in a crossover design trial with two 42-d periods (28 d adaptation, 14 d sampling) and were fed a ration with hay, corn, and soyhulls with HMTBa (0.1%, DM basis) or without (CON). Whole rumen contents (WRC), rumen solids (RS) and rumen liquid (RL) samples were taken 3 h pre- and post-feeding on d 29 of each period. Samples were analyzed for bacteria sequencing. Additional rumen fluid, collected 3 h post-feeding was used to measure gas production using the ANKOM gas production system and alfalfa hay as substrate. On d 36 of each period, the effect of HMTBa supplementation on dry matter and NDF digestibility of alfalfa and grass hays were evaluated using in situ methods. In vitro gas production at 24 h was greater (P = 0.002) in rumen fluid from steers fed HMTBa than CON. Supplementing HMTBa increased DM digestibility of alfalfa hay at 4 (P = 0.001) and 12 h (P = 0.025) of incubation and increased (P = 0.013) that of grass hay at 24 h of incubation. In situ NDF digestibility was greater at 12 h of incubation for alfalfa hay (P = 0.005) and 24 h of incubation for grass hay (P = 0.006) with HMTBa supplementation. Feeding steers HMTBa tended (P = 0.074) to increase Proteobacteria in RS and (P = 0.039) Actinomycetales in RL. Supplementing HMTBa resulted in Ruminococcaceae accounting for a greater proportion of bacteria at the family level in RL (P = 0.048), RS (P = 0.054) and WRC (P = 0.037). Furthermore, feeding steers HMTBa increased (P = 0.028) Ruminococcus in WRC, and tended to increase in RL (P = 0.054) and RS (P = 0.052). Greater population of Ruminococcus sp. was detected in WRC (P = 0.022), RL (P = 0.031), and RS (P = 0.025) in HMTBa fed steers. In conclusion, supplementing HMTBa increases fibrolytic bacterial species, resulting in a greater gas production in vitro, and greater in situ DM and NDF digestibility of selected hays.