Exercise affects serum levels of amino acids and their metabolites, with important metabolic consequences. Since vitamin D impacts skeletal muscle protein degradation, we hypothesised that it would also impact exercise-induced changes in serum amino acid levels and the serum levels of arginine metabolites, influencing the body's ability to synthesise NO. Accordingly, we analysed the effect of a single high-dose vitamin D supplementation on the serum levels of various amino acids in ultramarathon runners. Thirty-five male amateur runners were assigned to the supplemented group, administered 150,000 IU vitamin D in vegetable oil 24 h before the run (n = 16), or the control (placebo) group (n = 19). Blood was sampled 24 h before, immediately after, and 24 h after the run. Changes in the serum levels of some amino acids were distinct in the two groups. The asymmetric dimethyl arginine levels were significantly decreased immediately after the run and increased 24 h later and were not affected by the supplementation. The symmetric dimethyl arginine levels were increased after the run in both groups but were lower in the supplemented group than in the placebo group 24 h after the run. The dimethylamine levels increased significantly in the supplemented group as compared to the placebo group. In conclusion, vitamin D impacts exercise-induced changes in serum amino acids and methylated arginine metabolites.