Ultra-marathon (UM) running is an extreme endurance exercise. However, the mechanisms triggered with its practice remain unclear. While it is documented that strenuous physical activity activates immune responses and vitamin D plays a role in immune system suppression, data on the relationship between vitamin D status and cytokine profile in athletic populations are limited. To analyse the relative mRNA expression levels of selected pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-17, TNF-α), COX-2, vitamin D receptor and abundance of selected inflammatory microRNAs (Hsa-miR-21, -miR-146a, -miR-150, -miR-155, -miR-222, -miR-223) before and after a 100 km race in amateur runners in the presence or absence of vitamin D supplementation. Twenty runners aged 36-40years were divided into two groups: with and without vitamin D3 supplementation (10,000units daily). Blood samples were collected before and 12 h after the UM. The mRNA expression levels of selected cytokines, and in peripheral blood and abundance of serum exosomal miRNAs were investigated using q-RT-PCR. After UM, the significant up-regulation of and hsa-miR-155 and down-regulation of were observed in the group with vitamin D supplementation. In its absence, hsa-miR-155 and -miR-223 were significantly up-regulated. Additionally, a reverse correlation was observed between expression level and abundance of hsa-miR-155 and -miR-223 in both groups. No statistical differences were noted when the other miRNAs and genes were examined in the groups and at the time points. The UM-induced mRNA expression pattern of pro-inflammatory cytokines could be influenced by vitamin D supplementation and/or miRNA.