The genus Acorus is a perennial hydrophyte used as a medicinal and aromatic plant. Acorus calamus and A. gramineus were cultured for in vitro investigation of growth rate, shoot formation and root formation on the media containing different kinds and concentrations of plant growth regulators. In A. calamus and A. gramineus, the seeds were germinated and the germination rate was almost 100%. Then, the seedlings were subcultured for the experiment on the MS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar at different kinds and concentrations of plant growth regulators, auxins and cytokinins. The best shooting rate was obtained with about 100% on MS media containing BA (6-benzyl amino purine) or TDZ (N-phenyl-N′-[(1,2,3-thidiazol-5-yl) urea]). The number of developed shoots was significantly increased at 4 mg·L−1 BA with 5.4 shoots or 1 mg·L−1 TDZ with 11.0 shoots than control with 0.1 shoots in A. calamus. The A. gramineus showed slow growth rate and low shoot proliferation rate than A. calamus in general. In A. gramineus, the shoot formation was best in 4 mg·L−1 BA with 2.7 shoots or 1 mg·L−1 TDZ with 3.9 shoots than control with 0.9 shoots, respectively. Kinetin treatments brought 100% rooting rate, which were higher than that of BA and TDZ. Root formation was the best on the media containing 1 mg·L−1 NAA and 0.5 mg·L−1 NAA in A. calamus and A. gramineus, respectively. The plantlets were transferred to the greenhouse and survival rate was 95% after acclimatization.