Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) fixes atmospheric carbon dioxide into bioavailable sugar molecules. It is also well known that a kinetic isotope effect (KIE; CO2 carbon atoms) accompanies the carboxylation process. To describe the reaction and the KIE α, two different types of molecular dynamics (MD) simulations (ab initio MD and classical MD) have been performed with an Own N-layered Integrated molecular Orbitals and molecular Mechanics (ONIOM)-hybrid model. A channel structure for CO2 transport has been observed during the MD simulation in RubisCO, and assuming the reaction path from the inlet to the product through the coordinate complex with Mg2+ , simulations have been performed on several molecular configuration models fixing several distances between CO2 and ribulose-1,5-bisphosphate along the channel. Free energy analysis and diffusion coefficient analysis have been evaluated for different phases of the process. It is confirmed that the isotopic fractionation effect for CO2 containing either 13 C or 12 C would appear through the transiting path in the channel structure identified in RubisCO. The estimated isotope fractionation constant was quite close to the experimental value. © 2020 Wiley Periodicals, Inc.