Most land plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. These fungi differentiate essential functional structures called arbuscules in root cortical cells from which mineral nutrients are released to the plant. We investigated the role of miR393, a microRNA that targets several auxin receptors, in AM root colonization. Expression of the precursors of the miR393 was down-regulated during mycorrhization in three different plant species: Solanum lycopersicum (Solanaceae), Medicago truncatula (Fabaceae) and Oryza sativa (Poaceae). Treatment of S. lycopersicum, M. truncatula and O. sativa roots with concentration of synthetic auxin analogs that did not affect root development, stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter for auxin response, was preferentially expressed in root cells containing arbuscules. Finally, overexpression of miR393 in root tissues resulted in downregulation of auxin receptor genes (TIR1, AFB) and in under-developed arbuscules in all three plant species. These results support the conclusion that miR393 is a negative regulator of arbuscule formation by hampering auxin perception in arbuscule-containing cells.