Vitamin D and its analogs are known for their role in the development of breast cancer and in immunomodulation. Our previous studies have shown the pro-metastatic effect of calcitriol and tacalcitol (PRI-2191) in young mice bearing 4T1 breast cancer and the anti-metastatic effect in aged ON ariectomized (OVX) mice. Therefore, the aim of our work was to characterize Th17 cell population in young and aged OVX mice bearing 4T1 tumors treated with calcitriol and PRI-2191. The expression of genes typical for Th17 cells was examined in splenocytes, as well as splenocytes differentiated with IL-6 and TGF-beta to Th17 cells (iTh17). Expression of genes encoding vitamin D receptor (Vdr) and osteopontin (Spp1) as well as the secretion of IL-17A were evaluated in iTh17 cells. PRI-2191 treatment increased the expression of Rora and Rorc transcription factors, Il17a, Il17re and I121 in iThl7 cells from young mice. In aged OVX mice this effect was not observed. Increased expression was observed in the case of Vdr and Spp1 genes in iThl7 cells from young mice treated with PRI-2191. What is more, in young mice treated with PRI-2191 the secretion of IL-17A to the culture media by iTh17 cells was increased, whereas in aged OVX mice a significant decrease was noted. Increased expression of Spp1 in young mice treated with PRI-2191 may enhance the differentiation of Th17 cells.