A medium supplement for high-yield culture of anaerobes is disclosed. The medium supplement includes N-acetylhexosamine, L-aspartic acid, L-cysteine and cobalamin. A culture medium including the supplement and a culture method using the culture medium are also disclosed. It is possible to provide an innovative method which is capable of achieving high-concentration culture of anaerobes that are difficult to culture in high yield. The method is cost-effective, and in particular, is capable of culturing large amounts of fastidious aerobes suitable for use in food and pharmaceutical applications.