At rest, administration of the short-chain fatty acid acetate suppresses fat oxidation without affecting carbohydrate utilization. The combined effect of increased acetate availability and exercise on substrate utilization is, however, unclear. With local ethics approval, we studied the effect of ingesting either sodium acetate (NaAc) or sodium bicarbonate (NaHCO3) at a dose of 4 mmol·kg-1 body mass 90 min before completing 120 min of exercise at 50% VO2peak. Six healthy young men completed the trials after an overnight fast and ingested the sodium salts in randomized order. As expected NaAc ingestion decreased resting fat oxidation (mean ± SD; 0.09 ± 0.02 vs. 0.07 ± 0.02 g·min-1 pre- and post-ingestion respectively, p < .05) with no effect upon carbohydrate utilization. In contrast, NaHCO3 ingestion had no effect on substrate utilization at rest. In response to exercise, fat and CHO oxidation increased in both trials, but fat oxidation was lower (0.16 ± 0.10 vs. 0.29 ± 0.11 g·min-1, p < .05) and carbohydrate oxidation higher (1.67 ± 0.35 vs. 1.44 ± 0.22 g·min-1, p < .05) in the NaAc trial compared with the NaHCO3 trial during the first 15 min of exercise. Over the final 75 min of exercise an increase in fat oxidation and decrease in carbohydrate oxidation was observed only in the NaAc trial. These results demonstrate that increasing plasma acetate concentration suppresses fat oxidation both at rest and at the onset of moderate-intensity exercise.