Abstract: A plantlet regeneration protocol was developed for Pinus thunbergii. The procedure involves 3 steps, axillary buds induction, buds growth and root induction. Axillary buds induction was found to be influenced by the concentration of 6-benzyladenine (6-BA) and alpha-naphthylacetic acid (NAA) in the induction medium as well as the age of the seedling from which the explant was taken. Of the various 6-BA and NAA combination tested, modified Gresshoff and Doy’s medium (GD) with 4 mg/L 6-13A and 0.05 mg/L NAA proved to be the most effective, and the right stage of seedling was 22 ~ 28 d. During the next 5 weeks of culture on the same 6-BA-free but with 0.1 mg/L NAA medium multiple shoots elongated from the buds. The medium containing GA3 did not support buds growth. Elongated shoots were transferred to half-strength WPM medium supplemented with 0.2 mg/L NAA for root initiation and 14 days later transferred to half-strength WPM medium without any hormone for root development. After 1 month, 23.3 ~ of the shoots had formed adventitious roots.