Penicillium marneffei, the only known dimorphic and pathogenic species in the genus of Penicillium, is responsible for severe to deadly infection in immunocompromised patients. In this study, P. marneffei was able to infect the greater wax moth Galleria mellonella. The increasing inoculum doses of P. marneffei cells resulted in greater larval mortality, and the larval mortality rate also depended on the incubation temperature after P. marneffei infection and different P. marneffei strains. Moreover, the phagocytosis of hemocytes to P. marneffei was investigated, and it showed that the phagocytosis was increasing during the infection. These results demonstrated that G. mellonella can be effectively used to facilitate the in vivo study of P. marneffei infection and hemocytes are the key component of the larvae's immune defenses.