Key points• Rapid exchange of metabolites like glucose and lactate between different cell types is crucial for energy supply to the brain.• Carbonic anhydrase 2 (CAII) enhances lactate transport in mouse cerebellar and cerebral astrocytes.• Enhancement of transport activity is independent of the enzyme's catalytic function, but requires binding of CAII to the C-terminal tail of the monocarboxylate transporter MCT1.• CAII could enhance lactate flux by acting as a 'proton collecting antenna' for MCT1.• By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide neurons with an increased supply of energy substrate.Abstract Rapid exchange of metabolites between different cell types is crucial for energy homeostasis of the brain. Besides glucose, lactate is a major metabolite in the brain and is primarily produced in astrocytes. In the present study, we report that carbonic anhydrase 2 (CAII) enhances both influx and efflux of lactate in mouse cerebellar astrocytes. The augmentation of lactate transport is independent of the enzyme's catalytic activity, but requires direct binding of CAII to the C-terminal of the monocarboxylate transporter MCT1, one of the major lactate/proton cotransporters in astrocytes and most tissues. By employing its intramolecular proton shuttle, CAII, bound to MCT1, can act as a 'proton collecting antenna' for the transporter, suppressing the formation of proton microdomains at the transporter-pore and thereby enhancing lactate flux. By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide the neurons with an increased supply of energy substrate. Abbreviations 4-MI, 4-methylimidazole; aCSF, artificial cerebrospinal fluid; AE1, anion exchanger isoform 1; BCA, bichionic acid; CA, carbonic anhydrase; Car2, carbonic anhydrase II coding gene; CD147, cluster of differentiation 147; Co-IP, co-immunoprecipitation; EAAT, excitatory amino acid transporter; GFAP, glial fibrillary acidic protein; GST, glutathione S-transferase; gp70, glycoprotein 70; MCT, monocarboxylate transporter; m/z, mass-to-charge ratio; NBCe1, electrogenic sodium bicarbonate cotransporter isoform 1; NHE1, sodium/hydrogen exchanger isoform 1; PDB, protein data bank; PLA, proximity ligation assay.