A single bout of exercise can potentiate the effect of insulin on skeletal muscle glucose uptake via activation of the AMPK-TBC1 domain family member 4 (TBC1D4) pathway, which suggests a positive correlation between AMPK activation and insulin sensitization. In addition, prolonged fasting in rodents is known to upregulate and thereby synergistically enhance the effect of exercise on muscle AMPK activation. Therefore, fasting may potentiate the insulin-sensitizing effect of exercise. In the present study, we mimicked exercise by in situ muscle contraction and evaluated the effect of a 36-h fast on muscle contraction-induced insulin sensitization. Male Wistar rats weighing 150-170 g were allocated to either a 36-h fasting or feeding group. The extensor digitorum longus (EDL) muscles were electrically contracted via the common peroneal nerve for 10 min followed by a 3-h recovery period. EDL muscles were dissected and incubated in the presence or absence of submaximal insulin. Our results demonstrated that acute muscle contraction and 36 h of fasting additively upregulated AMPK pathway activation. Insulin-stimulated muscle glucose uptake and site-specific TBC1D4 phosphorylation were enhanced by prior muscle contraction in 36-h-fasted rats, but not in fed rats. Moreover, enhanced insulin-induced muscle glucose uptake and Akt phosphorylation due to 36 h of fasting were associated with a decrease in tribbles homolog 3 (TRB3), a negative regulator of Akt activation. In conclusion, fasting and prior muscle contraction synergistically enhance insulin-stimulated TBC1D4 phosphorylation and glucose uptake, which is associated with augmented AMPK pathway activation in rodents.NEW & NOTEWORTHY In this study, we revealed that 36 h of fasting additively upregulated acute muscle contraction-induced AMPK pathway activation in rats. Besides, fasting and muscle contraction synergistically enhanced insulin-stimulated site-specific TBC1D4 phosphorylation and glucose uptake, which was associated with augmented AMPK pathway activation. These results contribute to understanding the regulation of muscle insulin sensitivity.