Background The aim of this study was to explore the correlation between serum vitamin D levels in couples undergoing in-vitro fertilisation (IVF) and normal fertilisation process. Methods Between March 2016 and March 2017, we performed a prospective cohort study at an academic reproductive medicine centre to measure serum 25-hydroxyvitaminD (25-OHD) levels of 1232 couples before controlled ovarian stimulation. Generalized linear regression and binary multivariate logistic regression were employed to assess whether 25-OHD levels in men and women correlated with normal fertilisation rates and low fertilisation rate (LFR). Results Serum 25-OHD levels in women were classified into three groups: Group A, less than 10%; Group B, between 10 and 90%; and Group C, greater than 90%. Using generalized linear regression, we observed that female 25-OHD levels were related to normal fertilisation rates. Adjusted normal fertilisation rates from Group A to Group C in women were 59.50, 62.72, and 66.13%, respectively (P = 0.007). After binary logistic regression analysis, for women, compared with Group C, the ORs for LFR were 4.814 in Group A (95% CI, 1.266-18.309, P = 0.021) and were 3.204 in Group B (95% CI, 0.949-10.812, P = 0.061). Male 25-OHD levels were not related to the probability of low fertilisation rate (P > 0.05). Conclusions Circulating 25-OHD concentrations in women appear to be associated with normal fertilisation rates and low fertilisation rates in IVF cycles, but not in men. A further randomized controlled trial with vitamin D supplementation is needed to demonstrate whether female vitamin D levels exert an effect on the normal fertilisation process.