Cytosolic fumarase, a key enzyme for the accumulation of fumaric acid in Rhizopus oryzae, catalyzes the dehydration of L-malic acid to fumaric acid. The effects of carbon-nitrogen ratio on the acid production and activity of cytosolic fumarase were investigated. Under nitrogen limitation stress, the cytosolic fumarase could keep high activity. With the urea concentration decreased from 2.0 to 0.1 g l⁻¹, the cytosolic fumarase activity increased by 300% and the production of fumaric acid increased from 14.4 to 40.3 g l⁻¹ and L-malic acid decreased from 2.1 to 0.3 g l⁻¹. Cytosolic fumarase could be inhibited by substrate analog 3-hydroxybutyric acid. With the addition of 3-hydroxybutyric acid (50 mM) in the fermentation culture, fumaric acid production decreased from 40.3 to 14.1 g l⁻¹ and L-malic acid increased from 0.3 to 5.4 g l⁻¹.