Background: Sphingosine-1-Phosphate (S1P) has recently emerged as a key player in skeletal muscle metabolism through actions to specific kinases (SKs) and coupled-protein G receptors (S1PRs). Aim: Here we sought to determine the effect of short-term of S1P administration on physical performance and mitochondrial biogenesis markers in the skeletal muscle of mice. Methods: 40 male mice were divided in four groups: Wild-Type, Placebo, Exercise and Exercise plus intramuscular S1P (20 mg/kg per day) during 3 consecutive days. Thereafter, the animals performed an incremental exercise until exhaustion. Results: We demonstrate a relation between S1P and physical exercise in response to markers of mitochondrial biogenesis. Immunoblotting analysis showed an increase in phosphorylation of AMPK and AKT in response to S1P administration. The placebo group ran significantly less in comparison to S1P and Exercised groups. Conclusions: These preliminary findings suggest that physical exercise and S1P may enhance oxidative metabolism in the skeletal muscle and performance in mice.