The antidepressant venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is present in surface waters downstream of wastewater treatment plants. We previously showed that zygotic venlafaxine deposition alters larval behavior in zebrafish (Danio rerio), but the mechanisms were unknown. Here we tested the hypothesis that venlafaxine disrupts central serotonergic development, leading to impaired behavioral responses in zebrafish larvae. This was tested by microinjecting embryos with venlafaxine immediately after fertilization and performing spatial distribution of serotonin immunoreactivity, as well as characterizing target genes involved in serotonin turnover in the zebrafish brain. We provide evidence that venlafaxine exposure reduces serotonin immunoreactivity and tyrosine hydroxylase-positive cell populations in specific larval brain regions, and this corresponded with reduced larval activity observed in the drug-exposed group. Lowered serotonin was not due to either reduced synthesis or increased breakdown capacity. However, co-injection of serotonin alongside venlafaxine in embryos recovered brain serotonin immunoreactivity, tyrosine hydroxylase-positive cell populations, and rescued venlafaxine-mediated behavioral changes. Overall, our results demonstrate for the first time that early life exposure to venlafaxine perturbs brain development, which may be due to reduced serotonin, leading to altered larval behavior in zebrafish.