The blood-brain barrier (BBB) physiologically isolates the brain from the blood and thus plays a vital role in brain homeostasis. Ion transporters play a critical role in this process by effectively regulating access of chemicals to the brain. Organic anion transporting polypeptides (Oatp's) transport a wide range of amphipathic substrates and are involved in efflux of chemicals across the vertebrate BBB. The anatomical complexity of the vascularized vertebrate BBB, however, creates challenges for experimental analysis of these processes. The Drosophila BBB is structurally less complex, facilitating measurement of solute transport. Here we investigate a physiological function for organic anion transporting polypeptide 58Dc in transporting small organic anions across the BBB. We made use of genetic manipulation, immunocytochemistry and molecular techniques to supplement a whole animal approach used to study the BBB. This whole animal approach makes use of microinjecting the traceable small organic anion fluorescein into the haemolymph. This research shows that the organic anion transporting polypeptide 58Dc is involved in maintaining a chemical barrier against fluorescein permeation into the brain. Oatp58Dc was found to be expressed in the perineurial and subperineurial glia as well as postmitotic neurons. We specifically targeted knockdown of Oatp58Dc expression in the perineurial and subperineurial glia. This revealed that expression of Oatp58Dc in the perineurial glia is sufficient to maintain the barrier against fluorescein influx into the brain. Our results show that Oatp58Dc contributes to maintaining a functional barrier against fluorescein influx past the blood-brain barrier into the brain.