The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett-Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors' optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l(-1) of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l(-1)), glucose (12.7 g l(-1)), soy peptone (10.9 g l(-1)), and ethanol (13.8 ml l(-1)) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l(-1), a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l(-1) PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.