Purpose <p>Vitamin D status has been recognized as an important factor in human health, and its relevance in the context of couples utilizing assisted reproductive technologies is of particular interest.</p> Methods <p>This study aimed to assess the levels of 25(OH)D by ELISA in the biological fluids (serum, semen, and follicular fluid) of 105 couples undergoing in vitro fertilization or intracytoplasmic sperm injection. Additionally, the association among 3 vitamin D receptor polymorphisms (Apa1, Fok1, and Taq1) and vitamin D status, as well as the clinical pregnancy rate after in vitro fertilization, were investigated.</p> Results <p>The results revealed a high rate of hypovitaminosis in the women’s group. Furthermore, a significant difference was observed in the fertilization rate (<i>P</i> = 0.007). Correlations were found between Apa1 polymorphism and the number of collected oocytes (<i>P</i> = 0.014), the number of embryos (<i>P</i> = 0.001), the number of 2 pronuclear (<i>P</i> = 0.001), and the frequency of clinical pregnancies (<i>P</i> = 0.046). Additionally, correlations were found between Taq1 SNP and the number of embryos (<i>P</i> = 0.046) and the serum vitamin D level (<i>P</i> = 0.042). The Fok1 polymorphism showed correlations with semen concentration (<i>P</i> = 0.044) and clinical outcome (<i>P</i> = 0.032).</p> Conclusion <p>These results suggest the importance of systemic vitamin D for women prior to pregnancy. The findings emphasize the potential benefits of vitamin D supplementation in women before pregnancy and call for cautious interpretation of data regarding the impact of vitamin D on pregnancy.</p>