Phthalates are industrial plasticizers and stabilizers commonly found in polyvinyl chloride plastic and consumer products, including food packaging, cosmetics, medical devices, and children's toys. Di-(2-ethylhexyl) phthalate (DEHP), one of the most commonly used phthalates, exhibits endocrine-disrupting characteristics and direct exposure leads to reproductive deficits and abnormalities in anxiety-related behaviors. Importantly, increasing evidence indicates that the impacts of DEHP exposure on reproduction and social behavior persist across multiple generations. In this study, we tested the hypothesis that transgenerational DEHP exposure alters anxiety-like behavior and neural gene expression in both male and female mice. Pregnant CD-1 mice were orally dosed daily with either tocopherol-stripped corn oil or DEHP (20 or 200 μg/kg/day; 500 or 750 mg/kg/day) from gestational day 10.5 until birth to produce the F1 generation. Females from each generation were bred with untreated, unrelated CD-1 males to produce subsequent generations. Behavior and gene expression assays were performed with adult, intact F3 males and females. Transgenerational DEHP exposure increased time spent in the open arm in the elevated plus maze for adult females (750 mg/kg/day lineage), but not males. In adult females, we observed a down-regulation of mRNA expression of estrogen receptor 1 in the 200 μg/kg/day and 500 mg/kg/day treatment lineages, mineralocorticoid receptor in the 200 μg/kg/day lineage, and dopamine receptor 2 in the 20 μg/kg/day and 750 mg/kg/day lineages. In adult males, we found an up-regulation of estrogen receptor 2 in the 20 and 200 μg/kg/day lineages, and dopamine receptor 1 in the 20 μg/kg/day and 750 mg/kg/day lineages. No hippocampal gene expression modifications were observed in response to treatment. These results implicate dose-specific transgenerational effects on behavior and neural gene expression in adult male and female mice.