In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodateGlomeromycotafungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development.SbtM1is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legumeLotus japonicus.A detailed expression analysis by RT-PCR revealed thatSbtM1, SbtM3, SbtM4and the more distantly relatedSbtSare all rapidly induced during development of arbuscular mycorrhiza, but onlySbtSandSbtM4are also up-regulated during root nodule symbiosis. Promoter-reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression ofSbtM1orSbtM3by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.