The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) and N-methyl-N'-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain.