Background: Berkleasmium sp. Dzf12, an endophytic fungus from Dioscorea zingiberensis, was a high producer of palmarumycin C13 with various bioactivities. In the present study, the experimental designs based on statistics were employed to evaluate and optimize the medium for palmarumycin C13 production in mycelia liquid culture of Berkleasmium sp. Dzf12. Results: Among various carbon and nitrogen sources, glucose, peptone and yeast extract were found to be the most favourable for palmarumycin C13 production based on the one-factor-at-a-time experiments. After Plackett-Burman test on the medium, glucose, peptone and yeast extract were further verified to be the most significant factors to stimulate palmarumycin C13 accumulation. These three factors (i.e., glucose, peptone and yeast extract) were then optimized through the experiments of central composite design (CCD) and analysis of response surface methodology (RSM). The optimized medium compositions for palmarumycin C13 production were determined as 42.5 g/l of glucose, 6.5 g/l of peptone, 11.0 g/l of yeast extract, 1.0 g/l of KH2PO4, 0.5 g/l of MgSO4 x 7H2O, 0.05 g/l of FeSO4 x 7H2O, and pH 6.5. Under the optimal culture conditions, the maximum palmarumycin C13 yield of Berkleasmium sp. Dzf12 was increased to 318.63 mg/l, which was about 2.5-fold in comparison with that (130.44 mg/l) in the basal medium. Conclusions: The results indicate that the optimum production of palmarumycin C13 in Berkleasmium sp. Dzf12 liquid culture can be achieved by addition of glucose, peptone and yeast extract with their appropriate concentrations in the modified Sabouraud medium.