The symbiotic basidiomycete Hebeloma cylindrosporum is a model fungal species used to study ectomycorrhizal symbiosis at the molecular level. In order to have a vital marker, we developed a green fluorescent protein (GFP) reporter system efficiently expressed in H. cylindrosporum using the sgfp coding region bordered by two introns fused to the saprophytic basidiomycete Coprinopsis cinerea cgl1 promoter. Expression of this reporter system was tested under different environmental conditions in two transformants, and glucose was shown to repress gfp expression. Such a reporter system will be used in plant–fungus interaction to evaluate sugar supply by the plant to the compatible mycorrhizal symbiont and to compare the expression of various genes of interest in the free-living mycelia, in the symbiotic (mycorrhizas) and the reproductive (fruit bodies) structures formed by H. cylindrosporum.