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Arbuscular mycorrhizas (AMs) have the ability to enhance drought tolerance of citrus, but the underlying mechanisms have not been clearly elucidated. Considering the strong association of cell membrane fatty acid (FA) unsaturation with plant drought tolerance, the present study hypothesized that AM fungi (AMF) modulated the composition and unsaturation of FAs to enhance drought tolerance of host plants. Drought-sensitive citrus rootstocks, trifoliate orange (Poncirus trifoliata) seedlings, were inoculated with AMF (Funneliformis mosseae) for 3 months and were subsequently exposed to drought stress (DS) for 8 weeks. Mycorrhizal seedlings exhibited better plant growth performance, higher leaf water potential and lower root abscisic acid concentrations under both well-watered (WW) and DS conditions. Arbuscular mycorrhiza fungus inoculation considerably increased root methyl oleate (C18:1), methyl linoleate (C18:2) and methyl linolenate (C18:3N3) concentrations under both WW and DS conditions, and root methyl palmitoleate (C16:1) concentrations under WW, while it decreased root methyl stearate (C18:0) levels under both WW and DS. These changes in the composition of FAs of mycorrhized roots resulted in higher unsaturation index of root FAs, which later aided in reducing the oxidative damage on account of lower concentration of malondialdehyde and superoxide radicals. The changes of these FAs were a result of AMF-up-regulating root FA desaturase 2 (PtFAD2), FA desaturase 6 (PtFAD6) and Δ9 FA desaturase (PtΔ9) genes under WW and PtFAD2, PtFAD6 and Δ15 FA desaturase (PtΔ15) genes under DS conditions. Our results confirmed that mycorrhization brought significant changes in root FA compositions, in addition to regulation of gene expression responsible for increasing the unsaturation level of FAs, a predisposing physiological event for better drought tolerance of citrus.