To establish an infection, fungal pathogens must recog- nize diverse signals from host surfaces. The rice blast fungus, Magnaporthe oryzae, is one of the best models studying host-pathogen interactions. This fungus recog- nizes physical or chemical signals from the host surfac- es and initiates the development of an infection struc- ture called appressorium. Here, we found that protein MoAfo1(appressorium formation, MGG_10422) was involved in sensing signal molecules such as cutin mono- mers and long chain primary alcohols required for ap- pressorium formation. The knockout mutant (ΔMoafo1) formed a few abnormal appressoria on the onion and rice sheath surfaces. However, it produced normal ap- pressoria on the surface of rice leaves. MoAfo1 localized to the membranes of the cytoplasm and vacuole-like organelles in conidia and appressoria. Additionally, the ΔMoafo1 mutant showed defects in appressorium mor- phology, appressorium penetration, invasive growth, and pathogenicity. These multiple defects might bepartially due to failure to respond properly to oxidative stress. These findings broaden our understanding of the fungal mechanisms at play in the recognition of the host surface during rice blast infection.