A substantial number of people at risk to develop type 2 diabetes could not improve insulin sensitivity by physical training intervention. We studied the mechanisms of this impaired exercise response in 20 middle-aged individuals at high risk to develop type 2 diabetes who performed a controlled eight weeks cycling and walking training at 80 % individual VO2peak. Participants identified as non-responders in insulin sensitivity (based on Matsuda index) did not differ in pre-intervention parameters compared to high responders. The failure to increase insulin sensitivity after training correlates with impaired up-regulation of mitochondrial fuel oxidation genes in skeletal muscle, and with the suppression of the upstream regulators PGC1α and AMPKα2. The muscle transcriptome of the non-responders is further characterized by an activation of TGFβ and TGFβ target genes, which is associated with increases in inflammatory and macrophage markers. TGFβ1 as inhibitor of mitochondrial regulators and insulin signaling is validated in human skeletal muscle cells. Activated TGFβ1 signaling down-regulates the abundance of PGC1α, AMPKα2, mitochondrial transcription factor TFAM, and of mitochondrial enzymes. Thus, the data suggest that increased TGFβ activity in skeletal muscle can attenuate the improvement of mitochondrial fuel oxidation after training and contribute to the failure to increase insulin sensitivity.