In this study, for better understanding of culture phases in L-lysine fermentation, we tried to analyze the difference between culture phases at protein level based on a global analysis of intracellular proteins using 2DE. In order to overproduce L-lysine using an auxotrophic mutant of Brevibacterium flavum, L-threonine concentration have to be maintained at a low level during fermentation because L-lysine synthesis in the strain is regulated by a concerted aspartate kinase inhibition by L-threonine and L-lysine. Therefore, practically, L-lysine fermentation starts with high L-threonine concentration for cell growth and at the same time of L-threonine starvation, L-lysine is then overproduced. During the fermentation, physiological states of cells are completely different before and after the L-threonine starvation, that is, during a cell growth phase and a L-lysine production phase. The cells obtained at these phases were analyzed by 2-DE. The results showed that clear difference in protein expression existed in cell growth phase and L-lysine production phase, which will give useful information for modeling and control of L-lysine fermentation process.