This study is to isolate and identify γ-amino butyric acid (GABA) producing lactic acid bacteria (LAB) from Makgeolii, traditional Korean rice wine and then establish the optimal culture conditions for GABA production. Sixty four LAB from Makgeolli were isolated according to the characteristics of the shape and color of the colony grown on MRS agar plate. The GABA production of the isolated strain cultured in MRS broth contained 1% MSG (mono-sodium glutamate) were determined and evaluated by TLC and HPLC analysis. Strain B-134 was selected for highest GABA production. From the analysis of 16S rRNA and glutamate decarboxylase B (gadB) gene sequences, strain B-134 was tentatively identified as a Lactobacillus plantarum subsp. plantarum B-134. Effects of culture parameters, including glutamic acid level, culture temperature, NaCl level, and pH on GABA production were investigated for culture optimization. The optimum culture condition for GABA production by B-134 were culture temperature of 37℃, pH of 5.7, NaCl content of 0% (w/v) and MSG content of 3% (w/v), which produced 25 mM of GABA during cultivation time of 48 hr. From these results, strain B-134 is expected to be utilized as useful microorganisms for GABA-enriched health beneficial food.