AbstractAssociation of bacteria with fungi is a major area of research in infection biology, however, very few strains of bacteria have been reported that can invade and reside within fungal hyphae. Here, we report the characterization of an endofungal bacterium Serratia marcescens D1 from Mucor irregularis SS7 hyphae. Upon re-inoculation, colonization of the endobacterium S. marcescens D1 in the hyphae of Mucor irregularis SS7 was demonstrated using stereo microscopy. However, S. marcescens D1 failed to invade into the hyphae of the tested Ascomycetes (except Fusarium oxysporum) and Basidiomycetes. Remarkably, Serratia marcescens D1 could invade and spread over the culture of F. oxysporum that resulted in mycelial death. Prodigiosin, the red pigment produced by the Serratia marcescens D1, helps the bacterium to invade fungal hyphae as revealed by the increasing permeability in fungal cell membrane. On the other hand, genes encoding the type VI secretion system (T6SS) assembly protein TssJ and an outer membrane associated murein lipoprotein also showed significant up-regulation during the interaction process, suggesting the involvement of T6SS in the invasion process.