In this study, itaconic acid production level has been significantly improved. We conclude that micro-titer plate cultivation is suitable for screening large numbers of fungal strains or comparison of various media. Copper is shown to be positively correlated with improved itaconic acid production. Although the role of copper in the metabolic pathway of organic acids is not yet clear, this new insight will allow further medium optimization. The performance of gpdA and hbd1 transformants in itaconic acid production was not better than the nontransformed control. The optimal conditions for itaconic acid production are clearly different from those optimal for citric acid or oxalic acid production. Further researchis required for itaconic acid production improvement.