A bifidogenic growth stimulator produced by Propionibacterium freudenreichii was purified, and its chemical structure was determined. We obtained 7.1 mg of a bifidogenic growth stimulator from 1738 g of lyophilized P. freudenreichii cells by silica gel column chromatography, Sephadex LH-20 column chromatography, and preparative HPLC. The mass of the bifidogenic growth stimulator was 217.037 (C11H7NO4) as determined by high resolution mass spectrometry. Various experimental analyses indicated that the chemical structure of the bifidogenic growth stimulator was 2-amino-3-carboxy-1,4-naphthoquinone.