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A homeobox that encodes a transcriptional factor containing a 180-bp DNA-binding motif, termed the homeodomain, is important in regulating morphogenesis in fungi and other eukaryotes. In this study, we functionally analyzed CoHox2, one of the 10 homeobox genes conserved in Colletotrichum orbiculare, the causal agent of cucumber anthracnose. The number of conidia formed by CoHox2 knockout mutants was reduced to 30% of that by the wild type. However, conidial morphology did not differ significantly between CoHox2 mutants and the wild type. The CoHox2 mutants formed typical appressoria on glass and caused anthracnose on cucumber. As shown in scanning electron micrographs, CoHox2 mutants also formed more setae than the wild type did. These findings suggest that CoHox2 positively regulates conidiation and negatively regulates seta formation. Expression experiments based on fluorescent mCherry protein demonstrated that conidia formed by setae of the wild type have a nucleus. In addition, setae of the wild type incubated on PDA for 7 days had acutely pointed tips and were melanized, whereas those of the wild type incubated on PDA for 13 days had germinated and hyaline hyphae had developed. Thus, setae of the wild type may function in the formation of conidia with a nucleus in the early stage of culture and hyphal growth at the later stage.