Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity.