Metformin-induced glycolysis and lactate production can lead to acidosis as a life-threatening side effect, but slight increases inblood lactate levels in a physiological range were also reported in metformin-treated patients. However, how metforminincreases systemic lactate concentrations is only partly understood. Because human skeletal muscle has a high capacity to pro-duce lactate, the aim was to elucidate the dose-dependent regulation of metformin-induced lactate production and the potentialcontribution of skeletal muscle to blood lactate levels under metformin treatment. This was examined by using metformin treat-ment (16–776μM) of primary human myotubes and by 17 days of metformin treatment in humans. As from 78lM, metformininduced lactate production and secretion and glucose consumption. Investigating the cellular redox state by mitochondrial respir-ometry, we found metformin to inhibit the respiratory chain complex I (776lM,P<0.01) along with decreasing the [NADþ]:[NADH] ratio (776lM,P<0.001). RNA sequencing and phospho-immunoblot data indicate inhibition of pyruvate oxidation medi-ated through phosphorylation of the pyruvate dehydrogenase (PDH) complex (39lM,P<0.01). On the other hand, in humanskeletal muscle, phosphorylation of PDH was not altered by metformin. Nonetheless, blood lactate levels were increased undermetformin treatment (P<0.05). In conclusion, thefindings suggest that metformin-induced inhibition of pyruvate oxidation com-bined with altered cellular redox state shifts the equilibrium of the lactate dehydrogenase (LDH) reaction leading to a dose-de-pendent lactate production in primary human myotubes.