Metformin is the most prescribed initial anti-hyperglycaemic medication for people with Type 2 Diabetes. Exercise also has many beneficial health effects and is often recommended concomitantly. Although exercise also improves glycaemia, this effect is inhibited when undertaken alongside metformin ingestion [1,2]. Metformin intake also appears to further inhibit the beneficial response to exercise, including ablating improvements in insulin sensitivity and skeletal muscle mitochondrial protein synthesis and respiration [3,4]. Therefore, we propose that the skeletal muscle transcriptional response to exercise is disrupted by metformin. In this study, we hypothesise that metformin alters the transcriptomic response to exercise, and that this plays a role in the metformin inhibition of the beneficial exercise response. To test this, we analysed skeletal muscle biopsies from a previous study [5]. In this study, seven healthy lean male participants completed one bout of single-leg exercise with a contralateral non-exercising control-leg after acute metformin/placebo supplementation in a crossover design. After an overnight fast, muscle biopsies from the vastus lateralis were obtained in both legs after 1 hr of rest. Thereafter, 1.5 g metformin/placebo was ingested together with a standardized breakfast, the following 4½ hr were spent lying on a bed, after 2 hr a second metformin dose of 1.5 g metformin/placebo was ingested, at 4½ hr a second needle biopsy in both legs was taken. Immediately after this, the participant performed one-legged knee extensor exercise for 40 min with an intensity of 80% PWL (peak work-load) and a third biopsy was taken from both legs. RNA sequencing analysis was performed on biopsy samples from the rest and exercised leg from metformin/placebo groups, after 1 hr of rest, after 4½ hr (after metformin/placebo supplementation), and after exercise. This analysis revealed that metformin supplementation inhibited the transcriptomic response to exercise by reducing the total number of differentially expressed genes in response to exercise. After exercise, 81 genes were upregulated in response to exercise in both the metformin and placebo trials. 53 genes were uniquely upregulated in the placebo trial, while only 17 genes were uniquely upregulated in the Metformin trial. Importantly, the transcription factor NR4A3 was upregulated in response to exercise during the placebo trial (adj.p=0.011), but not when participants consumed metformin (adj.p=0.234). In HSMM (Human skeletal muscle myotubes), metformin (10µM) did not change NR4A3 expression (RT-qPCR, 1.2-fold-change, p=0.536) compared to the control. As expected, the exercise mimetic Ionomycin (8µM) significantly increased NR4A3 expression (4.3-fold-change, p<0.001), while concomitant metformin incubation significantly reduced the relative NR4A3 response (2.4-fold-change, p=0.002). In summary, we propose that the NR4A3 response to exercise is inhibited by metformin and that this may be a mechanism inhibiting exercise training-induced adaptations and skeletal muscle insulin sensitisation during metformin treatment.