Our study demonstrated that the NAPDH oxidase gene MtRbohE is expressed in arbusculated cells and plays a role in arbuscule development. Plant NADPH oxidases, known as respiratory burst oxidase homologs (RBOH), belong to a multigenic family that plays an important role in the regulation of plant development and responses to biotic and abiotic stresses. In this study, we monitored the expression profiles of five Rboh genes (MtRbohA, MtRbohB, MtRbohE, MtRbohG, MtRbohF) in the roots of the model species Medicago truncatula upon colonization by arbuscular mycorrhizal fungi. A complementary cellular and molecular approach was used to monitor changes in mRNA abundance and localize transcripts in different cell types from mycorrhizal roots. Rboh transcript levels did not drastically change in total RNA extractions from whole mycorrhizal and non-mycorrhizal roots. Nevertheless, the analysis of laser microdissected cells and Agrobacterium rhizogenes-transformed roots expressing a GUS transcriptional fusion construct highlighted the MtRbohE expression in arbuscule-containing cells. Furthermore, the down regulation of MtRbohE by an RNAi approach generated an altered colonization pattern in the root cortex, when compared to control roots, with fewer arbuscules and multiple penetration attempts. Altogether our data indicate a transient up-regulation of MtRbohE expression in cortical cells colonized by arbuscules and suggest a role for MtRbohE in arbuscule accommodation within cortical cells.