Thymus decussatus is a perennial herbaceous endangered medicinal plant found on mountain tops within the Saint Katherine Protectorate, South Sinai, Egypt. A protocol for in vitro preservation of this rare and important medicinal plant via micropropagation was developed. Stem segment explants excised from one month in vitro germinated seedlings were cultured on Murashige and Skoog's Medium (MS medium) supplemented with different concentrations of 6-benzylaminopurine (BAP; 0.5, 1.0 and 1.5 mgl-1) and kinetin (KIN; 1.0, 2.0 and 3.0 mgl-1) for maximum shoot formation. MS medium supplemented with 1.0 mgl-1 KIN showed the best results after 4-5 weeks. For root formation, MS medium was fortified with different concentrations of 3-indole acetic acid (IAA; 0.5, 1.0 ,1.5 and 2.0 mgl-1) and naphthalene acetic acid (NAA; 0.5, 1.0 ,1.5 and 2.0 mgl-1). After 5-6 weeks, the highest root numbers were obtained on MS medium with 1.0 mgl-1 NAA. Plantlets with well-developed roots were successfully acclimated with 73% survival.