Respiratory-deficient mutants of Rhizopus oryzae (R. oryzae) AS 3.3461 were acquired by ultraviolet (UV) irradiation to investigate changes in intracellular NADH metabolic pathway and its influence on the fermentation characteristics of the strain. Compared with R. oryzae AS 3.3461, the intracellular ATP level of the respiratory-deficient strain UV-1 decreased by 52.7 % and the glucose utilization rate rose by 8.9 %; When incubated for 36 h, the activities of phosphofructokinase (PFK), hexokinase (HK), and pyruvate kinase (PK) in the mutant rose by 74.2, 7.2, and 12.0 %, respectively; when incubated for 48 h, the intracellular NADH/NAD(+) ratio of the mutant rose by 14.6 %; when a mixed carbon source with a glucose/gluconic acid ratio of 1:1 was substituted to culture the mutant, the NADH/NAD(+) ratio decreased by 4.6 %; the ATP content dropped by 27.6 %; the lactate dehydrogenase (LDH) activity rose by 22.7 %; and the lactate yield rose by 11.6 %. These results indicated that changes to the NADH metabolic pathway under a low-energy charge level can effectively increase the glycolytic rate and further improve the yield of L-lactate of R. oryzae.