We investigated the effects of MgSO₄ on L-lactic acid production by the Rhizopus oryzae strain RLC41-6 in order to improve the production rate and quality of L-lactic acid. We also studied the effects of MgSO₄ on intracellular lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) activity. Different concentrations of MgSO₄ were added to the fermentation medium before culture of R. oryzae at 36°C for 36 h. L-lactic acid concentration was determined by reverse phase high performance liquid chromatography, and the activity and intracellular expression of LDH and ADH were examined via polyacrylamide gel electrophoresis and kinetic methods. Although increasing MgSO₄ concentrations led to increased lactic acid production by R. oryzae, MgSO₄ had no effect on LDH expression or activity. Furthermore, MgSO₄ promoted mycelium growth and inhibited ADH activity. With an initial MgSO₄ concentration of 0.075%, maximum mycelium production of 36.8 g/L was achieved and L-lactic acid was the only fermentation product, at ~138 g/L with a 92% conversion rate. An appropriate concentration of MgSO₄ can inhibit the conversion of pyruvic acid into malic acid and fumaric acid as well as both ADH activity and alcohol synthesis, thereby enhancing the conversion of glucose into lactic acid in R. oryzae.