Autism is a neurodevelopmental disorder characterized by impairments in sociability and communication. Prenatal exposure to Chlorpyrifos has been associated with autism-like behaviors in preclinical models. Interest has grown in the gut-brain axis and the role of microbiota modulation through dietetic supplementation to reduce this ASD-like phenotype. This study examines the effects of prenatal CPF exposure in Wistar Rats and assesses the potential of gestational probiotic and vitamin D supplementation to mitigate these effects in offspring. CPF exposure significantly impaired sociability in adolescence, and supplementation did not reverse these deficits. However, in control animals, supplementation induced neurodevelopmental changes, including alterations in metabolic status, the pattern of expression of ASD-related genes, the regulation of oxytocin and vasopressin receptors, and the GABAergic system in the brain. Additionally, supplementation accelerated overall development, increased ultrasonic vocalization emission and modified the typical responses to social novelty. CPF exposure blocked most of these effects at both behavioral and molecular levels. While supplementation did not block CPF-induced impairments, CPF exposure altered the observed effects of supplementation in controls, possibly indicating shared molecular mechanisms. These findings highlight the need for further research into the safety of probiotic and vitamin D supplementation during pregnancy.