A process for producing an O-alkylated rapamycin derivative of high purity, i.e. a purity is not lower than 99%, is porivided. A process for producing an o-alkylated rapamycin derivative represented by the following general formula (1) is provided. The process includes the steps of reacting rapamycin with an alkyl triflate, purifying a reaction product, which has been obtained by the above reaction, with a normal phase chromatograph and further purifying a purified product, which has been purified with the normal phase chromatograph, with a reverse phase chromatograph wherein R represents alkyl, arylalkyl, hydroxyalkyl, alkoxyalkyl, acyloxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxycarbonylaminoalkyl, acylaminoalkyl, or aryl.
