STUDY QUESTION What is the influence of vitamin D deficiency on pregnancy rates among women undergoing IVF/ICSI and Day 5 (blastocyst stage) single embryo transfer (SET)? SUMMARY ANSWER Vitamin D deficiency results in significantly lower pregnancy rates in women undergoing single blastocyst transfer. WHAT IS KNOWN ALREADY Preliminary experiments have identified the presence of vitamin D receptors in the female reproductive system. However, results regarding the effect of vitamin D deficiency on clinical outcomes are conflicting. None of the previous studies adopted a SET strategy. STUDY DESIGN, SIZE, DURATION Serum vitamin D concentration was measured retrospectively in patients who underwent SET on Day 5. Overall 368 consecutive infertile women treated within a period of 15 months were included in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS All patients underwent ovarian stimulation for IVF/ICSI and Day 5 SET. Serum samples were obtained 7 days prior to embryo transfer and stored frozen at -20°C. Samples were collectively analyzed for their 25-OH vitamin D content. Vitamin D deficiency was defined as serum 25-OH vitamin D levels <20 ng/ml in accordance with the Institute of Medicine and the Endocrine Society clinical practice guidelines. MAIN RESULTS AND THE ROLE OF CHANCE Clinical pregnancy rates were significantly lower in women with vitamin D deficiency compared with those with higher vitamin D values (41 versus 54%, P = 0.015).Logistic regression analysis was performed to identify whether vitamin D deficiency is independently associated with clinical pregnancy rates after controlling for 16 potential confounding factors. According to our results vitamin D deficiency was independently associated with lower clinical pregnancy rates, odds ratios [ORs (95% confidence interval (CI) 0.61 (0.39-0.95)] for vitamin D deficiency (deficient versus non-deficient women), P = 0.030. Finally, even when restricting our analysis to women undergoing elective SET (274 patients), vitamin D deficiency was again independently associated with pregnancy rates [OR (95% CI) 0.56 (0.33-0.93), P = 0.024]. LIMITATIONS, REASONS FOR CAUTION Our results refer only to patients undergoing Day 5 SET. Although vitamin D deficiency appears to compromise pregnancy rates in this population, no guidance can be provided regarding a potential relationship between vitamin D deficiency and ovarian reserve or response to ovarian stimulation. WIDER IMPLICATIONS OF THE FINDINGS Vitamin D deficiency impairs pregnancy rates in women undergoing single blastocyst transfer. Future prospective confirmatory studies are needed to validate our results and examine the exact underlying mechanism by which vitamin D levels may impair pregnancy rates in infertile women undergoing IVF/ICSI. STUDY FUNDING/COMPETING INTEREST(S) None declared. © 2014 The Author.