Eucalyptus globulus is an important rapid-growing trees in Eucalypts that applied for agroforestry and tree improvement programs. It has been used as a source of pulp in paper industry, essential oil and honey production. Asexual propagation with conventional methods such as cutting and graft wasnot possible or obtained success was very low, because of hard proliferation of adventitious roots. Then this study was focused on micropropagation of Eucalyptus globulus through mature trees. Apical and axillary buds from adult elite trees in forests of northern Iran were collected in different seasons. The method for sterilization and regeneration was optimized by comparing different combination of plant growth regulators. The highest number of induced axillary shoots was obtained in 0.5 mgl-1 of BA plus 0.5 mgl-1 of Kin after four weeks. Elongated shoots excised at 1.5 cm in length were rooted in 1/4 strenght of macroelements in MS medium with 0.5 mgl-1 of IBA plus 0.5 mgl-1 of NAA and activated charcoal (2.5 gl-1). These plants were transferred in sterile soil at greenhouse condition and successfully established in the field after gradual acclimation.