An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoot tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3 ± 0.9 and 15.0 ± 0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8 ± 1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4 ± 0.6 shoots per explant) or 10.0 μM kinetin (10.9 ± 0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055 ± 77 mg and 34.2 ± 1.0 mm, respectively) were significantly higher than those derived from full-strength MS semi-solid medium (437.6 ± 17.3 mg and 15.4 ± 0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after hardening.