The effect of a-acetolactate decarboxylase (ALDC) on the diacetyl content of beer was studied. Diacetyl is known as an off-flavor compound in beer due to its buttery flavor. ALDC is known to reduce diacetyl by bypassing the diacetyl formation pathway. Herein, beer was brewed with two-row barley (Jinyang) and six-row barley (Dahyang) cultured in Korea with the addition of ALDC. The formation of diacetyl is reversely affected by free amino nitrogen, such as valine. ALDC (0.02, 0.04, and 0.06 unit/mL) was added to the samples, and the physicochemical properties of the resulting beer were analyzed to determine the quality. Diacetyl content was decreased in proportion to the enzyme concentration. The diacetyl content in samples made from Jinyang and Dahyang decreased by 25 and 15%, respectively. The diacetyl content in both was acceptable for microbrewery.