In this paper, the establishment of seed regeneration system of Dioscorea nipponica from medicinal plant was studied, and the differences in anatomical structure between leaf, entangled stems and rhizome of tissue culture seedlings and solid seedling were investigated. The results showed that: (1) 15 min and 18 min 2% NaClO were suitable for disinfection. The optimal medium for callus induction was MS+1.0 mg·L_-16BA+2.0 mg·L_-12,4D, and its induction rate was up to 83.33%. MS+2.0 mg·L_-16BA+0.2 mg·L_-1NAA, induction rate was 90.00% in the best medium for bud induction. The best culture medium hormones ratio was 1/2MS+ 0.5 mg·L_-1NAA, generating 13.2 roots, its rooting rate was up to 86.70%. The growth of tissue culture seedlings is vigorous, the leaves are dark green, and the stems are thick and long. (2) There were significant differences in the structure of leaf and entangled stems between tissue culture seedlings and solid seedlings. The leaves of tissue culture seedlings were thin and the main vascular bundles were small and P/S low. The diameter, area and number of vascular bundles of the entangled stems were smaller than those of the solid seedlings; the catheter/vascular bundle was 0.06, which was 1/2 of the seedlings. Culture seedling proportion of cortex is large, but the differentiation between thickangle tissue and thickwalled tissue is not complete, and there are only two ductile vascular bundles with extremely dispersed arrangement. The changes in the shape and structure of tissue culture seedlings were mainly adapted to the culture environment of high humidity, low light, constant temperature and closed culture bottle.