Intense selection for elite racing performance in the Thoroughbred horse (Equus caballus) has resulted in a number of adaptive physiological phenotypes relevant to exercise, however the underlying molecular mechanisms responsible for these characteristics are not well understood. Adaptive changes in mRNA expression in equine skeletal muscle were investigated by real-time qRT-PCR for a panel of candidate exercise-response genes following a standardised incremental-step treadmill exercise test in eight untrained Thoroughbred horses. Biopsy samples were obtained from the gluteus medius before, immediately after and four hours after exercise. Significant (P < 0.05) differences in gene expression were detected for six genes (CKM, COX4I1, COX4I2, PDK4, PPARGC1A, and SLC2A4) four hours after exercise. Investigation of relationships between mRNA and velocity at maximum heart rate (VHR_max) and peak post-exercise plasma lactate concentration ([La]T₁) revealed significant (P < 0.05) associations with post-exercise COX4I1 and PPARCG1A expression and between [La]T₁ and basal COX4I1 expression. Gene expression changes were investigated in a second cohort of horses after a ten month period of training. In resting samples, COX4I1 gene expression had significantly increased following training and, after exercise significant differences were identified for COX4I2, PDK4 and PPARGC1A. Significant relationships with VHRmax and [La]T₁ were detected for PPARGC1A and COX4I1. These data highlight the roles of genes responsible for the regulation of oxygen-dependent metabolism, glucose metabolism and fatty acid utilization in equine skeletal muscle adaptation to exercise.