One of the defining features of eusocial systems is the allocation of tasks between morphologically or behaviorally distinct individuals within closely related families of reproducing and non-reproducing individuals. Such morphologically and behaviorally distinct individuals are known as “castes”. Behaviorally, the divergence and maintenance of such distinct individuals is established based on the crosstalk between hormonal signaling pathways underlying caste-specific activity, and the interplay between these and neuronal activities initiating or suppressing caste-specific behaviors. Brain, the organ at the intersection of neuronal and hormonal axes, shows transcriptomic, epigenomic and gene-regulatory signatures strongly associated with castes-specific behaviors. However, due to the heterogeneity and plasticity of the brain, it is challenging to use bulk profiling approaches to fully dissect how different cell types in brain organize internal and external neurohormonal signaling to develop behavioral caste identity.Here, we reveal an unanticipated role of the blood-brain barrier (BBB) in regulating complex social behavior between “soldier” and “forager” worker castes in the ant Camponotus floridanus. Using single-cell RNA-sequencing (scRNA-seq) to profile ant brains, we find that a key hormone-degrading enzyme called juvenile hormone esterase (Jhe) is highly-localized to the BBB. We show that the differential expression of this gene in the BBB between two worker castes results in differing levels of juvenile hormone III (JH3) entering the brain associated with caste-specific behaviors. Expression of this Jhe in the BBB only emerges and begins to differ during the late pupal stage. Manipulation of levels of Jhe via dsiRNA injection reprograms the brain transcriptome between ant castes. Most strikingly, Jhe manipulation via a single dose of dsiRNA is able to reprogram foraging-oriented behaviors between worker castes for at least 10 days. Although ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ants. Our study thus uncovers a remarkable, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates a complex social behavior.