The cucumber anthracnose fungus Colletotrichum orbicuIare forms specialized cells called appressoria for host penetration. We identified a gene, FAM1, encoding a novel peroxin protein that is essential for peroxisome biogenesis and that associates with Woronin bodies (WBs), dense-core vesicles found only in filamentous ascomycete fungi which function to maintain cellular integrity. The fam1 disrupted mutants were unable to grow on medium containing oleic acids as the sole carbon source and were nonpathogenic, being defective in both appressorium melanization and host penetration. Fluorescent proteins carrying peroxisomal targeting signals (PTSs) were not imported into the peroxisomes of fam1 mutants, suggesting that FAM1 is a novel peroxisomal biogenesis gene (peroxin). FAM1 did not show significant homology to any Saccharomyces cerevisiae peroxins but resembled conserved filamentous ascomycete-specific Pex22-like proteins which contain a predicted Pex4-binding site and are potentially involved in recycling PTS receptors from peroxisomes to the cytosol. C. orbiculare FAM1 complemented the peroxisomal matrix protein import defect of the S. cerevisiae pex22 mutant. Confocal microscopy of Faml-GFP (green fluorescent protein) fusion proteins and immunoelectron microscopy with anti-Fam1 antibodies showed that Fam1 localized to nascent WBs budding from peroxisomes and mature WBs. Association of Fam1 with WBs was confirmed by colocalization with WB matrix protein CoHex1 (C. orbiculare Hex1) and WB membrane protein CoWsc (C. orbiculare Wsc) and by subcellular fractionation and Western blotting with antibodies to Fa ml and CoHex1. In WB-deficient cohex1 mutants, Fam1 was redirected to the peroxisome membrane. Our results show that Fam1 is a WB-associated peroxin required for pathogenesis and raise the possibility that localized receptor recycling occurs in WBs. IMPORTANCE Colletotrichum orbicuIare is a fungus causing damaging disease on Cucurbitaceae plants. In this paper, we characterize a novel peroxisome biogenesis gene from this pathogen called FAM1. Although no genes with significant homology are present in Saccharomyces cerevisiae, FAM1 contains a predicted Pex4-binding site typical of Pex22 proteins, which function in the recycling of PTS receptors from peroxisomes to the cytosol. We show that FAM1 complements the defect in peroxisomal matrix protein import of S. cerevisiae pex22 mutants and that fam1 mutants are completely defective in peroxisome function, fatty acid metabolism, and pathogenicity. Remarkably, we found that this novel peroxin is specifically localized on the bounding membrane of Woronin bodies, which are small peroxisome-derived organelles unique to filamentous ascomycete fungi that function in septal pore plugging. Our finding suggests that these fungi have coopted the Woronin body for localized receptor recycling during matrix protein import.