NMDA receptors (NMDARs) are glutamate-gated ion channels involved in excitatory synaptic transmission and in others physiological processes such as synaptic plasticity and development. The overload of Ca2+ ions through NMDARs, caused by an excessive activation of receptors, leads to excitotoxic neuronal cell death. For this reason, the reduction of Ca2+ flux through NMDARs has been a central focus in finding therapeutic strategies to prevent neuronal cell damage. Extracellular H+ are allosteric modulators of NMDARs. Starting from previous studies showing that extracellular mild acidosis reduces NMDA-evoked whole cell currents, we analyzed the effects of this condition on the NMDARs Ca2+ permeability, measured as "fractional calcium current" (Pf, i.e. the percentage of the total current carried by Ca2+ ions), of human NMDARs NR1/NR2A and NR1/NR2B transiently transfected in HeLa cells. Extracellular mild acidosis significantly reduces Pf of both human NR1/NR2A and NR1/NR2B NMDARs, also decreasing single channel conductance in outside out patches for NR1/NR2A receptor. Reduction of Ca2+ flux through NMDARs was also confirmed in cortical neurons in culture. A comparative analysis of both NMDA evoked Ca2+ transients and whole cell currents showed that extracellular H+ differentially modulate the permeation of Na+ and Ca2+ through NMDARs. Our data highlight the synergy of two distinct neuroprotective mechanisms during acidosis: Ca2+ entry through NMDARs is lowered due to the modulation of both open probability and Ca2+ permeability. Furthermore, this study provides the proof of concept that it is possible to reduce Ca2+ overload in neurons modulating the NMDAR Ca2+ permeability.