Background: Proficient crop production is needed to ensure the feeding of a growing global population. The association of bacteria with plants plays an important role in the health state of the plants contributing to the increase of agricultural production. Endophytic bacteria are ubiquitous in most plant species providing, in most cases, plant promotion properties. However, the knowledge on the genetic determinants involved in the colonization of plants by endophytic bacteria are still poorly understood.Results: In this work we have used a genetic approach based on the construction of fliM, pilX and eps knockout mutants to show that the motility mediated by a functional flagellum and the pili type IV, and the adhesion modulated by exopolysaccarides are required for the efficient colonization of rice roots by the endophyte Azoarcus sp. CIB. Moreover, we have demonstrated that expression of an exogenous diguanylate cyclase or phophodiesterase, which causes either an increase or decrease of the intracellular levels of the second messenger cyclic di-GMP (c-di-GMP), respectively, leads to a reduction of the ability of Azoarcus sp. CIB to colonize rice plants.Conclusions: Here we present results demonstrating the unprecedented role of the universal second messenger cyclic-di-GMP in plant colonization by an endophytic bacterium, Azoarcus sp. CIB. The involvement of bacterial motility and adherence mediated by the bacterial flagellum, pil i type IV and exopolysaccharides in the colonization of rice by strain CIB is also shown. These studies pave the way to further strategies to modulate the interaction of endophytes with their target plant hosts.