608-614Lactobacillus acidophilus belongs to probiotic microflora inhabiting human gut that provide beneficially enhances human health. Besides balancing the intestinal flora and inhibiting pathogenic microorganisms, the existence of L. acidophilus inside the intestine can restore gut flora following antibiotics treatments. However, usually microorganisms from lactic acid bacteria group are known as fastidious microorganism and naturally required complex nutrients to promote their cellular growth. Therefore, twelve reported cultivation media were screened for their capability to support cell growth of L. acidophilus. The most suitable medium was further optimized using response surface methodology (RSM) and Box-Behnken design to maximize cell growth of L.acidophilus. Using this statistical approach, about 2.5-fold increase in maximal cell dry weight was achieved (5.14 g L-1) compared to the original medium (2.05 g L-1).This increase was accompanied by a significant increase in cell growth rates as well. The new medium formulation composed of (g L-1): glucose, 50; yeast extract, 20.91; ammonium citrate, 3.42; citric acid, 0.5; KH2PO4, 1.5; MgSO4.7H2O, 0.4; MnSO4.7H2O, 0.05; sodium acetate, 1; tween 80, 1