Inorganic phosphate (Pi) metabolism in arbuscules of arbuscular mycorrhizal (AM) fungi is not well understood, although recent research has revealed that host plants absorb Pi around arbuscules with mycorrhiza-specific transporters. Therefore, we analysed the localisation of polyphosphate (polyP) and alkaline phosphatase (ALP) activity in arbuscules, which could be indicators of Pi metabolism. We developed a dual-labelling method for polyP and ALP activity, i.e. first labelling with fluorescent probes 4′,6-diamidino-2-phenyl-indole dihydrochloride (DAPI) and then labelling with enzyme-labelled fluorescence (ELF97). The dual-labelling method made it possible to observe polyP and ALP activity signals simultaneously in mycorrhizal roots. The dual-labelling method revealed that ALP activity was mainly observed in mature arbuscules where polyP was rarely observed. The expression of the AM fungal ALP gene was suppressed in the knockdown plants of an AM-inducible Pi-transporter, and there was much polyP in arbuscules that showed low ALP activity. These topological observations suggest that there may be some relationships between polyP metabolism and ALP activity in arbuscules, and that these are, in part, controlled by Pi uptake by plants via the AM-inducible Pi-transporter.