► It was favorable for industry due to low cost and high polygalacturonase activity. ► Aspergillus niger and Saccharomyces cerevisiae were co-cultured for PG production. ► Co-culture system could endure higher temperature for PG production. Catabolic repression in the synthesis of inducible enzymes by glucose, fructose, and intermediates of the glycolytic cycle has been observed in many microorganisms. In order to enhance the polygalacturonase (PG) production of Aspergillus niger GJ-2, Saccharomyces cerevisiae J-1 was inoculated to the medium at 12h of culture, which resulted in a significant improvement of PG production. It was also found that maximum PG activity of 512.7U/ml was obtained at 37°C in the mixed culture, which was nearly twofold higher than that of the culture without the inoculation of S. cerevisiae J-1.