This research was carried out at the Plant Tissue Culture Laboratory (PTCL) in Faculty of Environmental Agricultural Sciences, Arish University, Egypt during 2013 to 2016. The aim of this study was using tissue culture technique for the micropropagation of Origanum syriacum L. plant. One node cutting and shoot tip were cultured on Murashige and Skoog (MS), Nitsch and Nitsch (NN) and Gamborg (B5) media. The results showed that shoot tip cultured on the MS medium had the best combination for the establishment stage of mother plants. Shoots cultured on MS medium supplemented with0.50 mgl^-1 Kin and 0.05 mgl^-1NAA produced the highest shoots developed during multiplication stage. MS medium supplemented with 0.40 mgl^-1 thiamine achieved the best shoots development compared with the other additive treatments and control. Addition of 1 mgl^-1IBA to the full strength MS medium and 1.00 mgl^-1 IAA to the half strength MS had the highest number of main roots and root lengths. Hardening the produced rooted plantlets were done in a greenhouse in pots containing mixture of peatmoss, vermiculite and washed sand at the rate (1:1:1) and after adjustment of mixture pH. Plantlets were successfully acclimated with 93 % survive.