Deficiencies in iron and vitamin D are frequently observed in athletes. Therefore, we examined whether different baseline vitamin D3 levels have any impact on post-exercise serum hepcidin, IL-6 and iron responses in ultra-marathon runners. In this randomized control trial, the subjects (20 male, amateur runners, mean age 40.75 ± 7.15 years) were divided into two groups: experimental (VD) and control (CON). The VD group received vitamin D3 (10,000 UI/day) and the CON group received a placebo for two weeks before the run. Venous blood samples were collected on three occasions—before the run, after the 100 km ultra-marathon and 12 h after the run—to measure iron metabolism indicators, hepcidin, and IL-6 concentration. After two weeks of supplementation, the intervention group demonstrated a higher level of serum 25(OH)D than the CON group (27.82 ± 5.8 ng/mL vs. 20.41 ± 4.67 ng/mL; p < 0.05). There were no differences between the groups before and after the run in the circulating hepcidin and IL-6 levels. The decrease in iron concentration immediately after the 100-km ultra-marathon was smaller in the VD group than CON (p < 0.05). These data show that various vitamin D3 status can affect the post-exercise metabolism of serum iron.