Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by persistent social communication deficits and restricted, repetitive behaviours, with significant overlap in anxiety-related symptoms. Both genetic and environmental factors contribute to the development of ASD, with early-life stressors, such as maternal separation (MS), and exposure to neurotoxic agents, like valproic acid (VPA), being key environmental contributors. This study investigates the combined impact of maternal deprivation (MD) and postnatal VPA exposure on autism-like behaviours and neurodevelopmental outcomes in male and female rats. Rats exposed to MD from postnatal days 2 to 4 exhibited significant changes in social interaction and anxiety-like behaviours, with female rats being more sensitive to MD than males. Postnatal VPA exposure resulted in similar behavioural alterations, including increased anxiety and social impairment, aligning with previous findings of VPA-induced neurodevelopmental deficits. A combination of MD and VPA exposure exacerbated anxiety-like behaviours in females, indicating that early-life stress and environmental toxins can synergistically affect neurodevelopment. Our results further suggest that the impact of these exposures may differ between sexes, with females showing heightened sensitivity to both MD and VPA-induced stress. These findings provide valuable insights into the complex interactions between genetic, environmental and epigenetic factors in ASD pathophysiology. The study underscores the critical role of early-life stressors, such as MD, in exacerbating neurodevelopmental disorders, particularly when combined with neurotoxic environmental factors like VPA. The sex-specific differences observed in behavioural outcomes suggest the importance of considering biological sex in future ASD research and therapeutic strategies.