•This study analyzed how PAs were involved in AMF-enhanced drought tolerance of plants.•AM trifoliate orange had higher Put and Cad and lower Spd and Spm under drought.•Mycorrhizal plants had higher activity of PA catabolic enzymes and Put synthetase.•AM plants showed overexpression of PtSPMS, PtPAO, PtSOD, and PtCAT1 under DS.•AM plants represented down-regulated expression of PtCuAO under DS. Arbuscular mycorrhizas enhance the drought tolerance of host plants through several underlying mechanisms. Polyamines (PAs) are known to protect plant cells from the damages of drought by enhancing the antioxidant defense system, restricting ethylene synthesis, maintaining cell pH and ion homeostasis, preventing chlorophyll loss, and so on; concomitantly, the mechanism through which arbuscular mycorrhizal (AM) fungi modulate PA metabolism to enhance drought tolerance of plants remains unclear. A pot experiment was conducted to assess how an AM fungus, Funneliformis mosseae, affects root PA homeostasis, activities and gene expressions of PA-related synthesizing and degrading enzymes, hydrogen peroxide (H2O2, a PA metabolite) production, and antioxidant enzyme gene expressions in trifoliate orange (Poncirus trifoliata) exposed to drought stress (DS). AM seedlings showed higher growth traits, leaf water potential, two plasma membrane intrinsic protein aquaporin gene expressions, and chlorophyll concentrations than non-AM seedlings under well-watered (WW) and DS conditions. Mycorrhizal treatment induced higher putrescine and cadaverine but lower spermidine and spermine concentrations, with higher activity of PA catabolic enzymes (copper-containing diamine oxidase, CuAO; polyamine oxidase, PAO) and putrescine synthases (ornithine decarboxylaseby; arginine decarboxylase, ADC). Mycorrhizas up-regulated the expression of the spermine synthase gene, PtSPMS, under DS, and down-regulated the transcript levels of PA catabolic enzyme genes (PtCuAO1, PtCuAO2, PtCuAO6, and PtCuAO8) and PA synthase genes (PtADC1 and PtADC2) under DS. PtPAO1, PtPAO2, and PtPAO3 had higher expression levels in AMF-inoculated seedlings, as compared to non-AMF-inoculated seedlings, under DS, triggering reactive oxygen species-related signalling for stress responsiveness through low H2O2 levels by up-regulating the expression of PtMn-SOD, PtCu/Zn-SOD, and PtCAT1. This study demonstrated that mycorrhizas have the capacity to modulate PA metabolism to enhance the drought tolerance of plants.