Research question: Do serum vitamin D levels in men and women undergoing IVF/intracytoplasmic sperm injection (ICSI) affect embryo development and clinical outcomes? Design: A large retrospective observational cohort study conducted between March 2016 and March 2017; plasma 25-OHD levels were measured before ovarian stimulation in couples undergoing IVF/ICSI (including 1883 women and 1720 men). Results: Women were divided into four groups in terms of serum 25-OHD quartiles: <14.8 ng/ml (Q1), 14.8-19.6 ng/ml (Q2), 19.7-24.7 ng/ml (Q3) or >24.7 ng/ml (Q4). Statistically significant differences in top-quality embryo rates were found among different groups on Day 3 (P = 0.016) but not on Day 2 or Day 5/6. Specifically, the top-quality embryo rate on Day 3 was highest in Q2, a statistically significant difference compared with Q3 (P = 0.014). The multiple linear regression showed that serum 25-OHD levels in women were not significantly related to top-quality embryo rates on Day 2, Day 3 or Day 5/6. There were no differences in implantation, clinical pregnancy, miscarriage and live birth rates among the groups. Logistic regression revealed that there were no significant differences in the success of clinical pregnancy. In men, no significant association was found between vitamin D status and embryo development or clinical outcomes. Conclusions: In neither men nor women were vitamin D levels associated with embryo development in cleavage (Day 2 and Day 3) and blastocyst (Day 5/6) stage and clinical outcomes. This may be because the threshold of vitamin D affecting the reproductive process is relatively low; a multicentre study in a high-latitude region is needed.