Activation of aryl hydrocarbon receptors (AhRs) induces neuronal damage, but the mechanism by which this occurs is largely unknown. This study evaluated the effects of an AhR agonist, β-naphthoflavone, on apoptotic pathways in mouse primary neuronal cell cultures. β-Naphthoflavone (0.1–100 μM) enhanced caspase-3 activity and lactate dehydrogenase (LDH) release in neocortical and hippocampal cells. These data were supported at the cellular level with Hoechst 33342 and calcein AM staining. α-Naphthoflavone inhibited the action of β-naphthoflavone, thus confirming specific activation of AhRs. A high-affinity estrogen receptor (ER) antagonist, ICI 182,780, and a selective estrogen receptor modulator (SERM), tamoxifen, enhanced β-naphthoflavone-mediated apoptosis. Another SERM, raloxifene, and an ERα antagonist, methyl-piperidino-pyrazole, did not affect β-naphthoflavone-induced caspase-3 activity. However, they inhibited β-naphthoflavone-induced LDH release at a late hour of treatment, thus suggesting delayed control of AhR-mediated neuronal cell death. The apoptotic effects of β-naphthoflavone were accompanied by increased levels of AhRs, and these receptors colocalized with ERβ as demonstrated by confocal microscopy. These data strongly support apoptotic effects of AhR activation in neocortical and hippocampal tissues. Moreover, this study provides evidence for direct interaction of the AhR-mediated apoptotic pathway with estrogen receptor signaling, which provides insight into new strategies to treat or prevent AhR-mediated neurotoxicity.