Plantago major L., a species that originated in the Far East, has been used for medicinal aspects since ancient times. The technique for callus induction, growth, and plant regeneration from cultured young leaves and petiole explants of Plantago major was carried out. The best initiation and growth of calli was achieved on MS supplemented with 1 mgL-1 2,4-D and 0.5 mgL-1 BA. Shoot regeneration from the calli was obtained on MS medium containing 1 mgL-1 TDZ alone or combined with 0.5 mgL-1 -NAA (100 and 96%, respectively). Moreover, the optimum medium for shoot multiplication was MS medium comprising 5 mgL-1 BA, and root formation from the shoots was half-strength MS medium supplemented with 2 mgL-1 NAA. The regenerated plants were successfully transferred into pots containing a mixture of decayed straw: rice husk ashes, (1:1, v/v), sand: soil (1:1, v/v), soil, or sand and grown in nethouse. The phenotypes of regenerated plants were normal as a mother plant.