Optimization of the fermentation medium components for maximum gentamicin production by Micromonospora echinospora ATCC 15838 was carried out. Response surface methodology was applied to optimize the medium constituents. A 2 4 full-factorial central composite design was chosen to explain the combined effects of the four medium constituents, viz. starch, soyabean meal, K2HPO4, and CoCl 2 and to design a minimum number of experiments. A second order model was developed and fitted using least square method. The R 2 value of the model was 0.9723, which shows that model is best fit for the present studies. The results of analysis of variance and regression of a second order model showed that the linear effects of starch (p < 0.001697) and CoCl2 (P < 7.99E-13), and cross product effects of starch and soyabean meal (p < 0.029876) and soyabean meal and CoCl2 (P < 0.008909) were more significant, suggesting that these were critical variables having the greatest effect on the production of gentamicin in the production medium. The optimized medium consisting of 9 g/L starch, 3 g/L soyabean meal, 0.9 g/L K 2 HPO4, and 0.01 g/L CoCl2 predicted 850 mg/L of gentamicin which was almost 110% higher than that of the unoptimized medium. The amounts of starch, soyabean meal, and K2HPO4 required were also reduced with RSM.