The objectives of this study were to optimize fermentation conditons of anti-tumor active metabolites from marine penicillium sp. HGQ6. Based on the inhibition activity against stomach cancer cell BGC823, firstly, the initial medium was screened, then seven factors including peptone, sucrose, K2HPO4, FeSO4, pH, temperature and agitation were investigated by Plackett-Burman design, and the important variables including pH, temperature and K2HPO4 were optimized by using Response surface methodology (RSM). Experimental results showed that the optimum fermentation conditions were peptone 1%, sucrose 3%, K2HPO4 0.06%, FeSO4 0.001%, pH6.41, temperature 35°C and agitation 180 r·min-1. Under these optimal conditions, the actual inhibition ratio achieved 90.4%, which agreed with the predicted values. This indicated the good feasibility of Plackett-Burman design and RSM in the optimization of fermentation conditions for production of anti-tumor active substances from stain HGQ6.