The PGC-1α is a master regulator of mitochondrial biogenesis in skeletal muscle. The expression of PGC-1α-b mRNA via the alternative promoter plays an important role in exercise-dependent expression of PGC-1α gene. The goal of the study was to investigate a role of AMPK in regulation of PGC-1α gene expression via the alternative promoter in endurance-trained skeletal muscle. We investigated activation of AMPK and PGC-1α gene expression via the alternative promoter before and after acute endurance exercise, with or without administration of a single dose of metformin, well known AMPK activator. In order to improve metformin delivery to skeletal muscle, the exercise bouts were performed when the level of metformin in the blood was near maximal. On the other hand, to avoid exercise-induced activation of AMPK, we used low-intensity exercise. The study was approved by the Human Ethics Committee of the IBMP RAS and complied with the guidelines set forth in the Declaration of Helsinki. 9 amateur endurance-trained athletes Vo2max 56 (53-62) ml/min/kg] participated in this study. Each participant swallowed 2 g of metformin or placebo before exercise (45 min, 40% Vo2max). Biopsies from the vastus lateralis muscle were taken prior to and at 2 min, 4 h, and 8 h after exercise. Data are expressed as median and interquartile range. The Wilcoxon test was used to compare repeated measurements at level of significance P ≤ 0.05. In the experimental trial the plasma concentration of metformin prior to and just before termination of exercise was high (~ 1000 mg/l). The exercise intensity was low, and therefore did not induce lactate accumulation in the blood in the placebo trial. The phosphorylation level of AMPKThr172 did not change at 2 min after the exercise in either the metformin or placebo trials. However, ACCSer79/222 (the substrate of AMPK, i.e. an endogenous marker of AMPK activity) showed a 2.6-fold (P < 0.01) increase in phosphorylation level at 2 min after exercise in the metformin trial only. Post-exercise expression of PGC-1α gene via both the alternative and canonical promoters did not differ between trials. Lack of a metformin-induced increase in PGC-1α gene expression via the canonical promoter under increased AMPK activity is in accordance with recent findings showing that the canonical promoter in endurance-trained human skeletal muscle is constitutively expressed, and that its expression does not depend on the intensity of exercise or intensity-dependent activation of AMPK (Popov et al., 2015). Lack of a metformin-induced increase in PGC-1α gene expression via the alternative promoter does not confirm a role of AMPK in regulation of PGC-1α gene expression in endurance-trained human skeletal muscle.