Hydrophobin proteins originate from filamentous fungi, which are able to self-assemble at water-air interfaces. Hydrophobins have multiple functions in fungal growth and development. In this study, the function of the Trhfb3 gene encoding a class II hydrophobin was characterized in Trichoderma reesei. The null mutant of Trhfb3 presented a wettable phenotype and a significantly reduced conidial production compared with the parent strain. The ΔTrhfb3 mutant also exhibited less biomass formation than the parent strain. In addition, Trhfb3 was expressed on carbon sources that induce lignocellulytic enzymes, with the highest expression level detected on cellobiose. The results show that Trhfb3 has a role in vegetative growth and asexual development in T. reesei.