This experiment aimed to assess the impact of virginiamycin on in vitro gas production dynamics, rumen kinetics, and nutrient digestibility in beef steers fed a grain-based diet. 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 troughs. 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 virginiamycin (VM) administration at 0 (VM0), 180 (VM180), or 240 mg/d (VM240). During d 15 and 16 of each period, about 600 mL of rumen fluid and urine samples were collected before (0 h), and at 4, 8, 12, and 16 h after the morning feed (0730 h), rumen inoculum was used to take pH and redox potential measurements immediately after collection using a portable pH and redox meter, and subsamples were taken for volatile fatty acids (VFA) and NH3-N analyses, and urine samples were composited daily and analyzed for creatinine and purine derivatives (PD) content to estimate microbial crude protein flow. During the 4-h post-morning feed rumen collection, rumen inoculum was utilized to perform in vitro gas production measurements. Fecal samples were collected on d 16 of each period to estimate nutrient digestibility using acid detergent insoluble ash as an internal marker. Animals were considered the experimental unit for the statistical analyses, and periods and squares were included as random variables. The total and rate of gas production were similar among treatments (P ≥ 0.17). The second-pool (i.e., fiber) gas production increased linearly as VM inclusion increased (P = 0.01), with VM240 being greater compared to VM180 and VM0 (7.84, 6.94, and 6.89 mL, respectively). Ruminal pH linearly increased as VM increased, with VM240 being greater than VM0 and VM180 intermediate (5.90, 5.82, and 5.86, respectively; P = 0.03). The VFA concentrations did not differ (P ≥ 0.13), but the acetate-to-propionate ratio was the highest in VM240 (P = 0.005). Branched-chain VFA increased (P ≤ 0.03) while lactate concentrations decreased (P = 0.005) linearly with VM. The ruminal NH3-N concentration was the lowest in the VM0 (P = 0.006). The estimated absorbed PD, purine derivative to creatinine index, and microbial N flow increased linearly with VM (P ≤ 0.07). The provision of VM influenced rumen dynamics in a dose-dependent manner.