The primary cause of harmaline tremor, which is a model of essential tremor (ET) in animals, is excessive activation of olivocerebellar glutamatergic climbing fibers. Our recent study indicated that 5′-chloro-5′-deoxy-(±)-N6-(±)-(endo-norborn-2-yl)adenosine (5′Cl5′d-(±)-ENBA), a potent and selective adenosine A1 receptor (A1) agonist, inhibited harmaline tremor.The present study was aimed to evaluate the role of glutamatergic transmission system in 5′Cl5′d-(±)-ENBA tremorolytic action in the harmaline model in rats, by analyzing glutamate release in the motor nuclei of the thalamus and mRNA expression of glutamatergic neuron markers (vGlut1/2) in reference to the general neuronal activity marker (zif-268) in different brain structures. The extracellular glutamate level in the motor thalamus was evaluated by in vivo microdialysis and the vGlut1/vGlut2 and zif-268 mRNA expression was analyzed by in situ hybridization. The intensity of tremor was measured automatically using Force Plate Actimeters (FPAs).5′Cl5′d-(±)-ENBA (0.5 mg/kg) given 30 min before harmaline (30 mg/kg) decreased the harmaline-induced excessive glutamate release in the motor thalamus and reversed harmaline-induced molecular effects, such as elevation of the vGlut1 mRNA expression in the inferior olive (IO) and decrease in the motor cortex, as well as an increase of the zif-268 mRNA expression in the IO, motor thalamus and motor cortex. Moreover, 5′Cl5′d-(±)-ENBA reduced harmaline tremor by lowering its power in 9–15 Hz frequency band.Our findings show that A1 stimulation decreases glutamate release in the motor thalamic nuclei in the harmaline model of ET, suggesting that A1 receptors, especially in this structure, may be a potential therapeutic target in this disorder.