Peroxisomes are ubiquitous organelles of eukaryotic cells that fulfill a variety of biochemical functions, including beta-oxidation of fatty acids. Here, we report that an ortholog of the Saccharomyces cerevisiae peroxisome biogenesis gene PEX13 is required for pathogenicity of Colletotrichum orbiculare. CoPEX13 was identified by screening random insertional mutants for deficiency in fatty acid utilization. Targeted knockout mutants of CoPEX13 were unable to utilize fatty acids as a carbon source. Expression analysis using green fluorescent protein fused to the peroxisomal targeting signals PTS1 and PTS2 revealed that the import machinery for peroxisomal matrix proteins was impaired in copex13 mutants. Appressoria formed by the copex13 mutants were defective in both melanization and penetration ability on host plants, had thin cell walls, and lacked peroxisomes. Moreover, the concentration of intracellular glycerol was lower in copex13 appressoria than those of the wild type. These findings indicate that fatty acid oxidation in peroxisomes is required not only for appressorium melanization but also for cell wall biogenesis and metabolic processes involved in turgor generation, all of which are essential for appressorium penetration ability.