Two-dimensional clay materials possess superior thermal and chemical stability, and the intrinsic tubular channels in their atomic structure provide possible routes for proton penetration. Therefore, they are expected to overcome the lack of materials that can conduct protons between 100-500 °C. In this work, we investigated the detailed proton penetration mechanism across 2D clay nanosheets with different isomorphic substitutions and counterions using extensive ab initio molecular dynamics and metadynamics simulations. We found that the presence of negative surface charges can dramatically reduce the proton penetration energy barrier to about one-third that of the neutral case, making it a feasible choice for the design of next-generation high-temperature proton exchange membranes. By tuning the isomorphic substitutions, the proton conductivity of single-layer clay materials can be altered.