Previous studies have shown that the short-term intake of a high-fat diet (HFD) impairs glucose metabolism. In this study, we investigated the influences of pre-exercise HFD intake for 3 d on post-exercise glycogen repletion in skeletal muscle in ICR mice. Mice received either an HFD (57% kcal from fat, 23% kcal from carbohydrate; HFD group) or standard laboratory chow (13% kcal from fat, 60% kcal from carbohydrate; Con group) for 3 d before exercise. Mice performed treadmill running at 25 m/min for 60 min and were orally administered a glucose (2 mg/g body weight) solution immediately after and at 60 min after exercise. A negative main effect of pre-exercise HFD intake was observed for skeletal muscle glycogen concentration from the pre-exercise phase to 120 min of post-exercise recovery ( p <0.01). Blood glucose concentration in the HFD group was significantly higher than in the Con group at 120 min after exercise ( p <0.01). No significant difference was observed in plasma insulin concentration. There were no significant between-group differences in the phosphorylation state of Akt Thr308, AMPK Thr172, AS160 Thr642, or glycogen synthase Ser641 or in glucose transporter 4 protein levels during post-exercise recovery. Our results suggest that the intake of a pre-exercise HFD for 3 d affects post-exercise glycogen repletion in skeletal muscle without impairing the insulin signaling cascade.