Pyruvate dehydrogenase (PDH) is an important regulator of carbohydrate oxidation during exercise and its activity can be down-regulated by an increase in dietary fat. The purpose of this study was to determine the acute metabolic effects of differential dietary fatty acids on the activation of PDH in its active form (PDHa) at rest and at the onset of moderate-intensity exercise. University-aged male subjects (n=7) underwent two fat loading trials spaced at least 2 weeks apart. Subjects consumed ~300 g saturated (SFA) or n-6 polyunsaturated (PUFA) fat over the course of 5 hours. Following this, participants cycled at 65% VO₂ max for 15 min. Muscle biopsies were taken prior to and following fat loading and at 1 min exercise. Plasma free fatty acids increased from 0.15±0.07 to 0.540.19 mM over 5 hours with SFA and from 0.11±0.04 to 0.35±0.13 mM with n-6 PUFA, and were significantly lower throughout the n-6 PUFA trial. PDHa activity was unchanged following fat loading, but increased at the onset of exercise in the SFA trial, from 1.18±0.27 to 2.16±0.37 mmol/min/kg wet wt. This effect was negated in the n-6 PUFA trial (1.04±0.20 to 1.28±0.36 mmol/min/kg wet wt.). PDH kinase (PDK) was unchanged in both trials, suggesting that the attenuation of PDHa activity with n-6 PUFA was a result of changes in the concentrations of intramitochondrial effectors, potentially intramitochondrial NADH or Ca²⁺. Our findings suggest that attenuated PDHa activity contributes to the preferential oxidation of n-6 PUFA during moderate-intensity exercise.