Epidemiological studies indicate that developmental vitamin D (DVD) deficiency is prevalent in women of childbearing age. Prenatal ethanol exposure (PEE) is also a plausible risk factor for long-lasting consequences in offspring because of elevated rates of alcohol consumption in women of childbearing age and the large proportion of unplanned pregnancies. Furthermore, there is an increased likelihood that these two common exposures take place simultaneously. However, there is a gap in the literature focusing on their combined risk, and it is important to address the effects of the combination of DVD deficiency and PEE on brain development. There were 2 main aims of this study; first, to analyse whether maternal ethanol metabolism was altered in vitamin D-deficient mice. Second, we aimed to assess the effects of DVD deficiency and PEE on global changes in gene expression at birth, using hypothesis-generating, unbiased screens on neonatal whole brain tissue. The results showed that maternal vitamin D status did not interfere with the levels of alcohol or alcohol dehydrogenase in blood sera, suggesting that there was no effect of vitamin D deficiency on the metabolism of ethanol. With regards to gene expression, RNA-Seq analysis confirmed the effects of DVD and PEE on signalling pathways previously noted in the literature as vulnerable to these exposures. However, the combination group (DVD-PEE) had the highest number of dysregulated transcripts involved with neurodevelopmental processes, such as cell function and maintenance, cell assembly and organization, and cell signalling, despite no overlap of altered molecules between single hit exposures (DVD or PEE). These data suggest that the combined effects of DVD-PEE act upon independent pathways from those affected by DVD or PEE alone. Therefore, based on these studies in mice we suggest that maintenance of optimal vitamin D levels and abstinence from alcohol during pregnancy would reduce risk of later disruption to brain function and behaviour in the offspring.