An isolate of Streptomyces was found to be positive for the production of tacrolimus. HPLC analysis on C-18 column at flow rate of 0.5 ml/min samples gave retention time of 4.26 min as standard. Soyoil, soybean meal and L-lysine were selected as significant model factors by plackett burman (PB) design. Response surface methodology was employed to optimize these factors. A second-order quadratic model and response surface method showed that the optimum conditions (soyoil: 23.40 gm/L; soybean meal gm/L: 18.40; L-lysine: 1.00 gm/L) resulted in the improvement of tacrolimus production (235.5± 1.96 mg/L) as compared to the initial level (135.6± 2.56 mg/L). It was about 1.73 fold increase as compared with that using the original medium. The value predicted by the quadratic model was (236.3 mg/L). Analysis of variance showed a high coefficient of determination (R2) value of 0.9978 ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is first report on tacrolimus production using plackett burman design and response surface methodology in submerged fermentation.