The impact of eccentric exercise on mitochondrial function has only been poorly investigated and remains unclear. This study aimed to identify the changes in skeletal muscle mitochondrial respiration, specifically triggered by a single bout of eccentric treadmill exercise. Male adult mice were randomly divided into eccentric (ECC, downhill running), concentric (CON, uphill running) and unexercised control groups (n = 5/group). Running groups performed 18 bouts of 5 min at 20 cm.s-1 on an inclined treadmill (+ or - 15 to 20°). Mice were sacrificed 48 hours post exercise for blood and quadriceps muscles collection. Deep proximal (red) and superficial distal (white) muscle portions were used for high-resolution respirometric measurements. Plasma creatine kinase activity was significantly higher in ECC compared to CON group, reflecting exercise-induced muscle damage (p < 0.01). The ECC exercise induced a significant decrease in oxidative phosphorylation capacity in both quadriceps femoris parts (p = 0.032 in proximal portion, p = 0.010 in distal portion) in comparison with the CON group. This observation was only made for the NADH pathway using pyruvate+malate as substrates. When expressed as a flux control ratio, indicating a change related to mitochondrial quality rather than quantity, this change appeared more prominent in distal compared to proximal portion of quadriceps muscle. No significant difference between groups was found for the NADH pathway with glutamate or glutamate+malate as substrates, for the succinate pathway or for fatty acid oxidation. Our data suggest that ECC exercise specifically affects pyruvate mitochondrial transport and/or oxidation 48 h post exercise and this alteration mainly concerns the distal white muscle portion. This study provides new perspectives to improve our understanding of the mitochondrial adaptation associated with ECC exercise.