This study investigated the combined effects of exercise training and D‐allulose intake on endurance capacity in mice. Male C57BL/6J mice were fed either a control diet (Con) or a 3% D‐allulose diet (Allu) and further divided into the sedentary (Sed) or exercise training (Ex) groups (Con‐Sed, Con‐Ex, Allu‐Sed, Allu‐Ex, respectively; n = 6–7/group). The mice in the Ex groups were trained on a motor‐driven treadmill 5 days/week for 4 weeks (15–18 m/min, 60 min). After the exercise training period, all mice underwent an exhaustive running test to assess their endurance capacity. At 48 h after the running test, the mice in the Ex groups were subjected to run at 18 m/min for 60 min again. Then the gastrocnemius muscle and liver were sampled immediately after the exercise bout. The running time until exhaustion tended to be higher in the Allu‐Ex than in the Con‐Ex group (p = 0.08). The muscle glycogen content was significantly lower in the Con‐Ex than in the Con‐Sed group and was significantly higher in the Allu‐Ex than in the Con‐Ex group (p < 0.05). Moreover, exercise training increased the phosphorylation levels of adenosine monophosphate‐activated protein kinase (AMPK) in the muscle and liver. The phosphorylation levels of acetyl coenzyme A carboxylase (ACC), a downstream of AMPK, in the muscle and liver were significantly higher in the Allu‐Ex than in the Con‐Sed group (p < 0.05), suggesting that the combination of exercise training and D‐allulose might have activated the AMPK‐ACC signaling pathway, which is associated with fatty acid oxidation in the muscle and liver. Taken together, our data suggested the combination of exercise training and D‐allulose intake as an effective strategy to upregulate endurance capacity in mice. This may be associated with sparing glycogen content and enhancing activation of AMPK‐ACC signaling in the skeletal muscle.