We tested whether physiological doses of hydroxytyrosol (HT) may alter the mRNA transcription of key metabolic genes in exercised skeletal muscle. Two groups of exercise-trained Wistar rats, HTlow and HTmid, were supplemented with 0.31 and 4.61 mg/kg/d of HT, respectively, for 10 weeks. Another two groups of rats were not supplemented with HT; one remained sedentary and the other one was exercised. After the experimental period, the soleus muscle was removed for qRT-PCR and western blot analysis. The consumption of 4.61 mg/kg/d of HT during exercise increased the mRNA expression of important metabolic proteins. Specifically, 4.61 mg/kg/d of HT may upregulate long-chain fatty acid oxidation, lactate, and glucose oxidation as well as mitochondrial Krebs cycle in trained skeletal muscle. However, a 4.61 mg/kg/d of HT may alter protein translation, as in spite of the increment showed by CD36 and GLUT4 at the mRNA level this was not translated to higher protein content.