Based on the cyclization reaction mechanism of intermediate rifamycin oxazine in synthesis of rifampicin, effect of pH value on purity of rifamycin oxazine was investigated as well as influences of regulating pH on cyclization reaction temperature, cyclization reaction time, purity and yield of rifamycin oxazine and rifampicin.The best pH value was determined to be from 5 to 7, and under this condition side reaction can be restrained, purity and yield of rifamycin oxazine and rifampicin influence can be improved.