Rapamycin is an important natural macrolide antibiotic with antifungal, immunosuppressive and anticancer activity produced by Streptomyces hygroscopicus. In this study, a mutant strain obtained by ultraviolet mutagenesis displayed higher rapamycin production capacity compared to the wild-type S. hygroscopicus ATCC 29253. To gain insights into the mechanism of rapamycin overproduction, comparative metabolic profiling between the wild-type and mutant strain was performed. A total of 86 metabolites were identified by gas chromatography-mass spectrometry. Pattern recognition methods, including principal component analysis, partial least squares and partial least squares discriminant analysis, were employed to determine the key biomarkers. The results showed that 22 potential biomarkers were closely associated with the increase of rapamycin production and the tremendous metabolic difference was observed between the two strains. Furthermore, metabolic pathway analysis revealed that amino acids metabolism played an important role in the synthesis of rapamycin, especially lysine, valine, tryptophan, isoleucine, glutamate, arginine and ornithine. The inadequate supply of amino acids, or namely "nitrogen starvation" occurred in the mutant strain. Subsequently, the exogenous addition of amino acids into the fermentation medium of the mutant strain confirmed the above conclusion, and rapamycin production of the mutant strain increased to 426.7 mg/L after adding lysine, approximately 5.8-fold of that in the wild-type strain. Finally, the results of real-time PCR and enzyme activity assays demonstrated that dihydrodipicolinate synthase involved with lysine metabolism played vital role in the biosynthesis of rapamycin. These findings will provide a theoretical basis for further improving production of rapamycin.