The allelopathic substance lepidimoide (Lp), which exhibits multiple functions in the growth and development of plants, was produced by Colletotrichum sp. AHU9748 from okra polysaccharide. Okra polysaccharide has the repeating structure (1 -> 4)-O-alpha-(D-galactopyranosyluronic acid) (1 -> 2)-O-alpha-L-rhamnopyranose in its hexasaccharide repeating unit of its main chain. To determine the enzymes essential for Lp production, the supernatant of a culture broth was fractionated by repeated column chromatographies to identify two serial fractions responsible for Lp production and non-Lp production by measuring Lp production together with P-galactosidase (P-gal), rhamnogalacturonan lyase (RG-lyase) and acetylesterase (AE) activities, which we hypothesized to be necessary for Lp production from the structure of Lp. We confirmed the presence of these three enzymatic activities in the highest-Lp-producing fraction. The addition of purified RG-lyase to fractions producing no or a small amount of Lp demonstrated that P-gal and RG-lyase activities are necessary for Lp production. The N-terminal amino acid sequences of the three separated proteins on SDS-PAGE confirmed the presence of enzymes identical to P-gal, RG-lyase and AE in the Lp-producing fractions.