Exercise-induced inflammation can influence iron metabolism. Conversely, the effects of vitamin D3, which possesses anti-inflammatory properties, on ultramarathon-induced heart damage and changes in iron metabolism have not been investigated. Thirty-five healthy long-distance semi-amateur runners were divided into two groups: one group received 150,000 IU of vitamin D3 24 h prior to a race (n = 16), while the other group received a placebo (n = 19). Serum iron, hepcidin (HPC), ferritin (FER), erythroferrone (ERFE), erythropoietin (EPO), neopterin (NPT), and cardiac troponin T (cTnT) levels were assessed. A considerable effect of ultramarathon running on all examined biochemical markers was observed, with a significant rise in serum levels of ERFE, EPO, HPC, NPT, and cTnT detected immediately post-race, irrespective of the group factor. Vitamin D3 supplementation showed a notable interaction with the UM, specifically in EPO and cTnT, with no other additional changes in the other analysed markers. In addition to the correlation between baseline FER and post-run ERFE, HPC was modified by vitamin D. The ultramarathon significantly influenced the EPO/ERFE/HPC axis; however, a single substantial dose of vitamin D3 had an effect only on EPO, which was associated with the lower heart damage marker cTnT after the run.