Abstract Virginiamycin (VM) possesses antimicrobial properties due to its blocking of protein synthase in Gram-positive bacteria, allowing it to reduce lactic acidosis and the incidence of liver abscesses in ruminants. Ruminal acidosis is a common metabolic disorder that affects feedlot and dairy cattle and occurs when the supply of organic acids from fermentation exceeds its absorption and degradation, accumulating acid content in the rumen. The objective of this study was to evaluate the effects of three different doses of VM administration on in vivo and in vitro ruminal digestion kinetics of beef steers consuming a high-grain diet [metabolizable energy (ME): 2.99 Mcal/kg; Crude Protein (CP): 15.2 % dry matter basis (DM)]. Nine ruminally cannulated British-crossbred steers (596 ± 49 kg) were assigned to this experiment. Animals were housed in three pens (n = 3/pen) equipped with a Calan gate feed system and water trough. Pens were enrolled in a 3×3 Latin square design containing three periods of 16 d, and a 5-d washout interval between periods. Dietary treatments consisted of VM administration at 0 mg/d (VM0), 180 mg/d (VM180), and 240 mg/d (VM240). During d 15 and 16 of each period, about 600 mL of rumen fluid was collected before (0 h) and at 4, 8, 12, and 16 h relative to the morning feed (0730 h) pH and redox potential (Eh) measurements were taken immediately after collection using a portable pH and redox meter, and subsamples were taken for volatile fatty acids (VFA), and NH3-N analyses. During the 4-h post-morning feed rumen collection, rumen inoculum was utilized to perform in vitro gas production (IVGP) measurements. All statistical procedures were performed using SAS software where steer was considered the experimental unit, and period and square were included as random. Acetate, propionate, and total VFA did not differ among treatments (P ≥ 0.50), whereas butyrate increased linearly (P = 0.033) as the VM dose increased. Acetate:propionate ratio did not differ among treatments (P = 0.273). Lactate concentration decreased linearly (P = 0.027) as the VM dose increased; likewise, pH increased linearly (P = 0.019) as the VM dose increased. Branched-chain VFA and NH3-N concentrations increased linearly (P ≤ 0.056) as the VM dose increased. The total and rate of gas production were similar among treatments (P ≥ 0.161). However, second-pool gas production increased linearly as VM inclusion increased (P = 0.023). The in vitro neutral detergent fiber digestibility did not differ among treatments (P = 0.984). The provision of VM altered the rumen dynamics in a dose-dependent manner. Animals consuming high-grain diets will likely promote rumen health through a more stable pH and fermentation profile.