In this study, a dynamic and equilibrium modeling of water distillation process for 18O isotope separation with multi-component distillation approach was studied. The mathematical model of this process was derived in order to obtain the equilibrium concentrations using isotopic exchange reactions through statistical methods based on stochastic exchanges and probability of molecular collisions. In this modeling, to calculate the liquid-vapor equilibriums, the separation factor (α) or volatility was considered as a function of column temperature. Then equations of this model were solved by numerical techniques in Matlab programming and distributions of isotopic water components were obtained in the distillation column under total reflux conditions. The simulation results showed that the separation factor has a significant effect on the 18O isotope separation as the H-labeled light components increased in the condenser above the column; the HD and D-labeled heavy components increased in the boiler due to the difference of the separation factor. Then experimental results of 18O in the condenser above the column and the reboiler from a semi-industrial plant were compared with the simulation results, which it showed a good conformity between the modeling and pilot results.