The mechanism of base-catalyzed hydrogen/deuterium exchange (HDX) of the non-labile hydrogens in an aromatic alkylpyridinium cation has been investigated in the present work. Although HDX reaction is not expected in non-labile hydrogens in general, non-labile hydrogens at the α-position of a pyridinium ring can occur in the base condition. Here, we precisely investigated the detailed kinetics of HDX at the hexadecylpyridinium cation in the methanol-d (CH₃OD) solvent with a base catalyst using electrospray ionization-mass spectrometry (ESI-MS). The numbers of exchanged hydrogens were counted by the series of time-dependent mass spectra during the HDX reaction, and the temperature-dependent reaction rates was determined under various base concentrations. The HDX reaction become faster as the concentration of the base catalyst increased, confirming the base-catalyzed mechanisms for HDX reaction. Temperature-dependent rate constants were used to evaluate the apparent activation energy using Arrhenius equation, and the dependence of the reaction order on the base concentration was also examine. Interestingly, the concentration of the base catalyst show a complicated relationship with the reaction order, deviating from the single-order relationship. Furthermore, the evaluated activation energy is largely different from the theoretical prediction which assuming a single-step base catalyzed HDX mechanism. This suggest that the detailed mechanism of the HDX reaction is not a simple single-step based catalyzed process, but may include multiple reaction steps in which surrounding solvents are involved as well. Further ab initio theoretical calculations are underway to reveal the exact mechanism in this HDX process.