Vitamin D has been utilized gestationally in both animal models of autism and human studies with promising efficacy. Robust meta-analyses in autism have linked the vitamin D receptor (VDR) gene, which stands out as one of the few consistently surviving meta-analyses in autism research. This suggests a potential role for vitamin D in the etiology or modulation of autism spectrum disorders. Our study aims to investigate the effects of prenatal vitamin D3 supplementation on neurodevelopmental outcomes in Wistar rat offspring. We focus on indicators of autistic-like behaviors (such as ultrasonic vocalizations and Crawley's test), as well as brain gene expression (via RT-qPCR) and metabolomic profiles. We aim to determine whether gestational interventions utilizing vitamin D3 supplementation alone can mitigate neurodevelopmental alterations observed in this context. Surprisingly, our results indicate that vitamin D3 supplementation alone induces changes in the aforementioned variables, suggesting a potential positive impact on neurodevelopment. However, these alterations were ultimately associated with disruptions in healthy control subjects. Given the widespread use of nutritional supplements containing vitamin D3 in contemporary populations, further exploration of the safety implications of such interventions is imperative.