The present study focused on improving docosahexaenoic acid (DHA) production by Schizochytrium sp. through N-methyl-N-nitro-N-nitrisiguanidine treatment coupled with ultraviolet radiation based on the metabolic pathway analysis. The activity of glucose-6-phosphate dehydrogenase of the mutant was higher than the parent strain, which indicated that the hexose monophosphate pathway of the mutant was strengthened, and more NADPH was thus produced. Also, the activities of malic enzyme and ATP-citrate lyase in the cell extract of the mutant were higher than the parent strain, which indicated that the screening method increased NADPH and acetyl-CoA supply in vivo effectively. Finally, in the batch culturing of the mutant, 34.84% higher lipid was accumulated with the cell dry weight at the same level compared with the parent strain. Moreover, the DHA percentage of the total fatty acids up to 56.22% was achieved using the mutant, which was 38.88% higher than the parent strain. When the cultures were maintained under appropriate conditions, the final DHA yield was 0.20 and 0.11 g/g dry biomass, for the mutant and parent, respectively.