Amiloride, a potassium sparing diuretic, is well known to interact with many ion transport systems and modulate the activity of several membrane receptors. However, relatively little information is available as to how amiloride affects membrane receptors of neurons in the brain areas. In the present study, we investigated the effects of amiloride on glycine-induced currents ( I Gly) in cultured neurons of rat inferior colliculus with whole-cell patch-clamp recordings. Amiloride itself did not activate any current across the neuronal membrane but it reversibly inhibited the amplitude of the I Gly in a reversible and concentration-dependent manner, with an IC 50 of 487.4 ± 25.3 μM ( n = 5). Amiloride shifted the concentration–response relationship to the right without changing Hill coefficient and without changing the maximum response of the I Gly. The pre-perfusion of amiloride produced an inhibitory effect on the I Gly. In addition, amiloride was shown with a voltage ramp protocol to significantly reduce the conductance induced by glycine but not to change the reversal potential of the I Gly. These results demonstrate that amiloride competitively inhibits the I Gly in rat inferior colliculus neurons by decreasing the affinity of glycine to its receptor. Our finding suggests that attention should be paid to the possible side effects of amiloride used as a drug on brain functions in the case of a defective blood–brain barrier and in the case of direct application of this drug into the cerebrospinal fluid for treatment of brain tumors.