Abstract: The effects of mole ratio of rifamycin oxazine to 1-amino-4-methyl piperazine, concentration, reaction time and temperature on the condensation of rifamycin oxazine were investigated. The condensation mechanism was studied and the related kinetic data were obtained, which provided guidance for the optimization of rifampicin synthesis. The results show that the optimal mole ratio of rifamycin oxazine to 1-amino-4-methyl piperazine is 1:2, and concentration has no significant effect on condensation. Both rifamycin oxazine conversion and rifampicin yield increase with the increase of reaction time or temperature. The condensation process includes two steps:(1) the rifamycin oxazine ring opens to form an intermediate and (2) the side chain of the intermediate is substituted by 1-amino-4-methyl piperazine to obtain rifampicin. The experimental data can be fitted by a second-order consecutive kinetic model. The activation energy of the ring opening process was 45.10 kJ·mol^-1 and that of the side chain substitution was 75.67 kJ·mol^-1.