The effect of L-valine (L-Val) on biosynthesis pathway of natamycin by Streptomyces natalensis HW-2 was investigated. The experimental results showed that the maximal yield of natamycin reached 1.83 g/L when the adding amount of L-Val was 0.5 g/L during the fermentation of Streptomyces natalensis HW-2 for 36 h, which was 84.85% higher than that of the control. The addition of L-Val resulted in a decrease of biomass and an increase of pH, and an improvement of glucose utilization. The activities of pyruvate kinase (PK), phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (PC) in intracellular enhanced, while that of citroyl synthetase (CS) decreased by 26.57%. Comparing with the control, the contents of pyruvic acid, oxaloacetic acid, acetyl-CoA in the broth increased by 80.50%, 53.28% and 47.19%, respectively. And, the contents of acetic acid, propionic acid and α-ketoglutaric acid increased by 16.98%, 10.65% and 15.40%, respectively. But, the content of citric acid decreased by 27.01%.