Whereas volatile fatty acid (VFA) production may be affected by product concentrations, this experiment assessed the effect of sodium acetate (NaOAc, 50 mM) addition on VFA and gas profile during in vitro fermentation. Rumen fluid samples (n = 16) with 1% timothy hay were incubated with or without 50 mM NaOAc addition. Tubes were equilibrated with a 50/50 gas mixture of CO₂ and N₂, and incubated at 39°C while shaking with 20-mL syringes attached to collect gases. VFA and gas production were measured at 0, 4, 16, 24, and 48 h. Means for treatments with acetate addition vs. control were analyzed using the model: y_{ij =} μ+A_i+ε_ij where, y_ij is the response, μ is the mean of the population, A_i is the effect of acetate (with or without 50 mmol addition), and ε_ij is the residual effect. The model was run by time. Means were compared using Student’s t-test and are reported when different (P < 0.05). Gas production is expressed in mmol/L of rumen fluid and VFA production is expressed as change in concentration (mM). Addition of acetate decreased gas production between 4 and 16 h (11.7 vs. 15.3 mmol/L of rumen fluid; SE ± 1.13). Acetate addition decreased acetate production at each interval: 0–4 h (−4 vs. 14 mM, SE ± 3.7); 4–16 h (11 vs. 26 mM; SE ± 4.5); 16–24 h (15 vs. 31 mM; SE ± 4.9); and 24–48 h (22 vs. 36 mM; SE ± 4.2). Acetate addition decreased propionate production from 0 to 4 h (4 vs. 6 mM; SE ± 0.5). Ratio of produced acetate:propionate (A:P) decreased from acetate addition for all intervals: 0–4 h (−0.8 vs. 2.4, SE ± 0.96); 4–16 h (1.1 vs. 2.3; SE ± 0.41); 16–24 h (1.1 vs. 2.3; SE ± 0.34); and 24–48 h (1.3 vs. 2.2; SE ± 0.23). The A:P ratio being close to 1 may be a result of acetate production being inhibited by NaOAc addition. NaOAc addition did not affect butyrate production, but decreased ratio of produced acetate:butyrate (A:B) for each interval: 0–4 h (−2.9 vs. 5.5; SE ± 2.6), 4–16 h (2.0 and 5.0; SE ± 0.89), 16–24 h (2.3 vs. 5.1; SE ± 0.74) and 24–48 h (3.0 and 5.2; SE ± 0.54). Acetate addition decreased total VFA production: 0–4 h (3 mM vs. 23 mM; SE ± 3.8) and 4–16 h (28 vs. 45 mM; SE ± 4.8). Addition of NaOAc to rumen fluid decreased subsequent production of acetate more than other VFA, and decreased gas production.