The Eucalyptus species are exotics in Iran, from Myrtaceae family and are planted widely in most countries for their fast growth, tolerant to poor soils and other excellent characteristics. Therefore, Eucalyptus seedling propagation is very important in Iran due to its role in extending forest plantation. Sexual and asexual propagation by seeds and cuttings causes problems due to high variable progenies and presence of rooting inhibitors in adult tissues, respectively. So, in vitro micropropagation techniques were used to prevent these problems. The aim of the study was to investigate the effects of growth regulators on shoot explants sampled from E. melliodora seedlings (internod segments).The experimental design was Randomized Complete Blocks (RCB) with nine treatments (BAP: 0.1, 9.6, 1.0 mgl-1 and 2ip: 0.5 and 0.6 mgl-1) and five replicates and five explants at each plot. The regenerated shoots were treated with GA3 (0.05 mgl-1), BAP and 2ip to increase their length growth. For rooting seven treatments of 2,4-D and NAA were applied at four concentrations (0, 0.1, 0.5 and 1.0 mgl-1) Although the effects of different growth regulators on regenerated shoot growth characteristics (length, number, weight and fresh shoots and bud numbers) were significant, but the effects of both growth regulators and GA3 were only significant on shoots length. The best treatments for explants shooting were: BAP+2ip (0.1, 0.5 mgl-1), BAP (0.1 mgl-1), BAP (0.6 mgl-1), 2ip (0.5 mgl-1) and 2ip (0.6 mgl-1), respectively. The best medium for rooting was the one without growth regulator where there was minimum callus and no aerial roots. Application of NAA concentration at 0.1 mgl-1 resulted in highest rooting. After climate hardening of the plantlets, they were transferred to soil and started a normal growth after a long period.