BackgroundThe maternal-offspring relationship, such as whether fetal sex influences maternal health, is essential to explore to advance prenatal and maternal health. While associations exist between fetal sex and maternal health outcomes, it is unclear whether these reflect a causal relationship. ObjectiveTo demonstrate that fetal sex can be randomly assigned to test the causal effect of fetal sex on maternal outcomes. MethodsHolstein dairy cows were stratified and randomized using sealed opaque envelopes to be artificially inseminated with either X- or Y-sorted bull semen until 40 cows became pregnant. Monthly body weight measurements were recorded, and an intravenous glucose tolerance test was performed 30 days before the expected calving day. The primary outcome was insulin area under the curve (AUC), and secondary outcomes were clearance rate, half-life, and AUC for glucose, insulin, and non-esterified fatty acid concentrations. An intention-to-treat (ITT) approach using multiple imputation was employed for primary analysis, and an as-treated (AT) approach was used for secondary analysis. ResultsWe demonstrated that we could successfully randomize the assignment of fetal sex to dams and test for causal effects of fetal sex on glucoregulatory outcomes using dairy cows as a model. Insulin AUC was not statistically different between groups (ITT p = 0.857, AT p = 0.874), and other outcomes were also not statistically different (p > 0.05). ConclusionWe demonstrated that causal effects of fetal sex on maternal outcomes can be causally tested in dairy cows. Our study did not provide statistical evidence to support an effect of fetal sex on maternal glucose-related outcomes.