Aims: To isolate an antagonist for use in the biological control of the phytopathogenic fungus Penicillium expansum and purify the antifungal component produced by the antagonist. Methods and Results: An antifungal strain HT16 was isolated from locusts, showing strong inhibition to Pen. expansum. Based on its in vitro effectiveness, HT16 was characterized as a strain of Paenibacillus polymyxa by phenotypic tests and 16S rDNA sequence analysis. It was found that the antifungal component HT16 secreted was only induced by Poria cocos sclerotium (PCS), and it remained active after sterilization at 121 degrees C for 15 min. The protein was purified by ammonium sulfate precipitation, heating process, and ultrafiltration using a 10 kDa cut-off membrane. The molecular weight of the purified antifungal protein, which was determined by mass spectrometry, was 4517 Da. Conclusions: A novel bacterial strain HT16 antagonistic to Pen. expansum was isolated from locusts and identified as Pae. polymyxa. The antifungal protein of 4517 Da was purified, and its production needed the inducer PCS in the fermentation medium. Significance and Impace of the Study: The antagonistic protein from Pae. polymyxa showed strong antifungal activity against phytopathogenic fungus Pen. expansum. This strain HT16 and the antifungal metabolite are therefore strong candidates for the biocontrol of phytopathogens in agriculture.