Abstract Rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischaemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischaemia, however the connecting link between the two is missing. Therefore we hypothesized that lactate might interact with TREK1 channels. In the present study, we show that lactate at ischaemic concentrations (15‐30 mM) at pH 7.4 increases TREK1 current in CA1 stratum radiatum astrocytes and causes membrane hyperpolarization. We confirm the intracellular action of lactate on TREK1 in hippocampal slices using monocarboxylate transporter blockers and at single channel level in cell‐free inside‐out membrane patches. The intracellular effect of lactate on TREK1 is specific since other monocarboxylates like pyruvate and acetate at pH 7.4 failed to increase TREK1 current. Deletion and point mutation experiments suggest that lactate decreases the longer close dwell time incrementally with increase in lactate concentration by interacting with the histidine residue at 328th position (H328) in the carboxy terminal domain of TREK1 channel. The interaction of lactate with H328 is dependent on the charge on histidine residue since isosteric mutation of H328 to glutamine did not show an increase in TREK1 channel activity with lactate. This is the first demonstration of a direct effect of lactate on ion channel activity. The action of lactate on TREK1 channel signifies a separate neuroprotective mechanism in ischaemia since it was found to be independent of the effect of acidic pH on channel activity. This article is protected by copyright. All rights reserved