While exercise performance deteriorates in hot environments, heat stress may contribute to exercise-induced adaptations in skeletal muscle. In this study, we assessed transcriptional profiles of equine skeletal muscle following 3 min of high-intensity exercise (at the speed eliciting their maximal oxygen uptake) in cool (Wet Bulb Globe Temperature [WBGT] 15°C) or hot (WBGT 30°C) conditions. Differential gene expression was identified using DESeq2 (false discovery rate cutoff: 0.05, minimal fold change: 1.5). At 4 h after exercise, RNA-seq identified 176 and 156 genes that were differentially expressed in the middle gluteal muscle in hot and cool conditions, respectively. Of these genes, 110 genes were altered in both conditions, whereas 66 genes were only responsive to exercise in the hot condition. Between the two environmental conditions, the expression of only one gene (KANK1) was higher in the hot condition compared with the cool condition. Pathway analysis revealed that the response to temperature stimulus was upregulated only after exercise in the hot condition. Although the overall transcriptional response to exercise was similar in both environmental conditions, our results provide insight into the molecular mechanisms of equine skeletal muscle adaptation to heat acclimation.