Moringa oleifera is the most important tree because each and every part of the plant hasrnnutritional and medicinal use. In Ethiopia studies related to this important plant are based onrnmicropropagation. Therefore, the main objective of this study was to optimize an in vitrornregeneration protocol for Moringa oleifera by using leaf as explants. Young leaves from in vitrornmultiplied shoots were excised, wounded perpendicular to midrib and cultured on MS mediumrnsupplemented with different concentrations of BAP in combination with NAA. For regenerationrnof shoot, the calli were cultured on MS medium supplemented with different concentrations ofrnBAP (0.0, 0.5, 1.0, 1.5, 2.0, mg/l) in combination with NAA (0.0, 0.5, 1.0, 1.5, 2.0 mg/l).rnMultiplication of regenerated shoots was done on MS medium supplemented with differentrnconcentrations of BAP (0.0, 0.5, 1.0, 1.5,2.0) in combination with IBA (0.0,0.5, 1.5, 2.5, 3.5 mg/lrn). Rooting was achieved by culturing well developed shoots in half strength MS mediumrncontaining 0.5 1.5, 2, 2.5, mg/l NAA or 0, 0.5, 1.0, 2.0, 3.0, 4.0 mg/l IBA. The highest (73.3%)rnpercentage of callus induction was obtained from the MS medium supplemented with 0.5mg/lrnBAP. When the callus transferred to the regeneration medium containing 0.5 NAA, it resulted inrnthe maximum regeneration rate of shoot (33.3%). Statistical analysis revealed that there wasrnsignificant difference among all treatments applied in both shoot multiplication and rootingrnexperiments. Maximum number of shoots per explants (3.13Â±0.73) was obtained on MS mediumrncontaining 1.0 mg/l BAP. The highest mean number of roots per shoot (9.60Â±0.86) was obtainedrnon MS medium containing 0.5mg/l IBA. After acclimatization, 90% plants survived inrngreenhouse. This protocol can be used for genetic improvement of this tree species.