Arbuscular mycorrhiza (AM) formed between AM fungi and the vast majority of higher plants is a widely spreading mutualistic symbiosis in the world. It has been proved that AM establishment starts with a chemical signal exchange between host plant roots and the fungi. Hydrogen peroxide (H₂O₂) is an important signal substance, which takes part in interaction among plants and many microorganisms. Does H₂O₂ also participate in the interaction between host plants and AM fungi? And we hypothesized that H₂O₂ may involve in the formation and colonization of AM. The purpose of the present research was to investigate the role of H₂O₂ in colonizing tobacco roots by arbuscular mycorrhizal (AM) fungi. Seedlings of tobacco cultivar CF90NF were inoculated with Glomus mosseae (G.m ). H₂O₂ contents, the effects of exogenous H₂O₂ on colonization percentage of tobacco by G.m, H₂O₂ scavenger and production inhibiter effects on H₂O₂ contents and fluorescence intensity in tobacco root and fungal hyphae were studied. The results showed that H₂O₂ burst in lateral roots of tobacco 20 days after inoculation, the earlier stage of colonization, at this time, fungal hyphae just began to contact with tobacco root and penetrate root cortical cell, exogenous H₂O₂ increased the colonization percentage of tobacco by G.m, H₂O₂ scavenger ascorbic acid (AsA) had adverse effect, decreased the colonization percentage; Fluorescence assay showed that G.m colonization induced intense H₂O₂ fluorescence in tobacco root, but AsA decreased H₂O₂ fluorescence intensity in tobacco lateral roots, and fungal hyphae. These results indicate that H₂O₂ have positive effect in symbiosis development between AM fungi and tobacco plants; PAO and POD is the main enzymes catalyzing the production of H₂O₂, we estimated the PAO and POD activities in tobacco roots, and the results showed that, when colonized with G.m, PAO and POD activities in lateral root of tobacco increased significantly, importantly, treatments with salicylhydroxamic acid (SHAM, POD inhibiter) and 1,12-diaminododecane (DADD, PAO inhibiter) decreased H₂O₂ fluorescence intensity in tobacco lateral roots, but had no significant effects on that in the fungal hyphae. The present study suggests that H₂O₂ may participate in tobacco-AM symbiosis, H₂O₂ be produced both in tobacco root and in fungal hyphae, PAO and POD take part in H₂O₂ production in tobacco root, and there should be other resource of H₂O₂ in AM fungal hyphae. While the interrelationship between H₂O₂ and the other signal substances, and their effects on the development of AM need to be further investigated with molecular techniques.