We report a successful and efficient plant regeneration system using mature embryo of barnyardgrass (Echinochloa crus). The effect of different sterilization procedures on explant culture and the effect of plant growth regulators on regeneration process and acclimatization of transplanting were assessed. It was found that EtOH+0.1%HgCl2 was optimal for explant sterilization. Essentially, dehulled and sterilized seeds were plated on callus induction and subculture medium composed of MS basal salts and DL vitamins plus 2.5 mg/L 2,4D, 300 mg/L glutamine, 〖JP1〗500 mg/L proline, 3% maltose, 0.8% (w/v) agar at pH 5.8. Differentiation was carried out on a differentiation medium MS with 50 mg/L〖JP+1〗 inositol plus 1.0 mg/L 6BA, 0.2 mg/L kinetin KT, 0.5 mg/L NAA, 0.25 mg/L IAA, 3% maltose, 0.8% (w/v) agar at pH 5.8. Plantlets ranging from 3 to 5 cm in length were transferred to the rooting media comprising 1/2MS basal salts. In vitro regenerated plantlets were washed to remove the traces of agar and then were transferred to pots containing nutritious soil and perlite (2〖DK1〗∶1, v/v), and the survival rate was over 90%. The protocol developed in this study will facilitate barnyardgrass generation and is useful for the production of herbicideresistant transgenic plants.