Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy, and the regulation of foetal growth across mammalian species. However, the temporal changes in maternal vitamin D status throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated. The abundance of 25(OH)D in maternal plasma was quantified by HPLC–MS/MS at gestational days (GD) 18, 30, 45, 60 and 90 (n = 5–11 gilts/GD). Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30 (P < 0.05). The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight, ovulation rate, mean litter weight, number of live foetuses, percentage prenatal survival, and sex ratio of the litter was assessed. Maternal 25(OH)D (P = 0.059) concentrations tended to be positively associated with percentage prenatal survival on GD60. On GD90, maternal 25(OH)D (P < 0.05) concentrations were inversely associated with gilt weight. Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90 (P < 0.05). This study has provided novel insights into temporal changes in maternal vitamin D status throughout gestation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight, percentage prenatal survival and percentage of male foetuses in the litter. Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species.