Perturbation of skeletal muscle homeostasis by exercise training promotes extracellular, contractile and metabolic modifications that increase the robustness of this tissue. These beneficial adaptations are a culmination of repeated, transient changes in post-exercise gene expression – coordination of which is heavily regulated by the activity of exercise-induced transcription factors. NR4A3 is one such transcription factor, identified as being responsive to aerobic exercise and recently hypothesised to be an important mediator of the adaptive response to exercise in rodents. Nevertheless, little is known regarding the role of NR4A3 in human skeletal muscle. A meta-analysis of publically available microarrays was performed to interrogate the response of NR4A3 after acute exercise, exercise training and chronic unloading of human skeletal muscle and its association with PPARGC1A expression. The contractile response of NR4A3 in cell culture was measured after electrical pulse stimulation (3 h at 40 V, 1 Hz and 2 ms) by RT-qPCR. The effect of siRNA silencing of NR4A3 on substrate flux and metabolic regulatory genes, with and without salbutamol treatment (20 µM), was quantified by Seahorse XF and RT-qPCR analysis, respectively. The impact of PPARGC1A viral overexpression on NR4A3 was also assessed by RT-qPCR. Primary human skeletal muscle myotubes from healthy donors were the model of study for all in vitro experiments and data is presented as mean ± SD. NR4A3 is upregulated in response to acute aerobic and resistance exercise but downregulated after chronic unloading in human skeletal muscle biopsies. Furthermore, NR4A3 and PPARGC1A expressions correlate in the acute (3 h) post exercise period, in sedentary cohorts only (r = 0.65, P = 0.003). In vitro, electrical pulse stimulation significantly increases NR4A3 relative to basal control (2.2 ± 0.9, P = 0.04). ~80% silencing of NR4A3 attenuates salbutamol-stimulated upregulation of glycolysis (3.7 ± 1.1 vs. 5.3 ± 1.1 mpH.min-1, P < 0.001). This affect may be associated with greater PDK4 in knockdown conditions (7.4 ± 6.1 vs. 1.7 ± 0.6 relative mRNA, P = 0.06). Viral overexpression of PPARGC1A results in ~1-fold increase of NR4A3 relative to GFP control (2.1 ± 1.5, P = 0.03), whilst PPARGC1A is unchanged by NR4A3 silencing. These data establish NR4A3 as a contraction-responsive, exercise-inducible transcription factor that influences metabolism in human skeletal muscle. NR4A3 may also operate downstream of PPARGC1A.