Carotenoids are precursors of several plant hormones and signaling molecules, which are involved in the establishment and the maintenance of arbuscular mycorrhizal (AM) symbiosis. Recently, we have identified zaxinone, a product of the rice carotenoid cleavage dioxygenase (OsZAS), as a novel natural metabolite and carotenoid-derived signaling molecule that is required for normal rice growth and development, affects arbuscular mycorrhizal symbiosis, and down-regulates strigolactone (SL) biosynthesis (Wang et al. 2019). A loss-of function mutant (oszas) showed decreased zaxinone content in roots, decreased root biomass, higher SL release and decreased mycorrhization level. To gain further insights into the role of zaxinone in mycorrhization, we generated OsZAS over-expressing lines under the control of 35S-promoter. These lines exhibited an increased mycorrhizal colonization level compared to WT. In order to understand whether zaxinone has a direct impact on the fungus during its pre-symbiotic phase, we tested the effect of this compound on spore germination of Gigaspora margarita, in comparison with the synthetic SL analog, GR24. To unravel the role of zaxinone during the colonization process, we determined the content of this compound and of SLs in oszas mutant and WT plants during a time course experiment. In their whole, the results suggest that, zaxinone, plays a crucial role in the development and establishing AM symbiosis in rice.