Many plant pathogenic fungi differentiate a series of highly specialized infection structures to invade and colonize host tissues. Especially at early stages of infection, the ratio of fungal to plant biomass is very low. To investigate cell-specific patterns of gene expression, it is necessary to purify the fungal structures of interest from infected plants. We describe here a method to isolate the biotrophic hyphae of Colletotrichum higginsianum from Arabidopsis leaves, based on a combination of pre-enrichment by isopycnic centrifugation followed by further purification by fluorescence-activated cell sorting. This protocol efficiently eliminates contamination by plant components and nontarget fungal cell-types. Moreover, the isolated cells remain alive, providing high-quality RNA for library construction. The method can be readily adapted for cell-specific transcriptome analysis in other plant–microbe interactions.