With the stem segments as explants,we studied the technique about the tissue culture for rapid propagation and in vitro bulbils regeneration system of Dioscorea opposita Thunb from Wen County,Henan Province.The results showed that:(1)The disinfection efficacy which combing 70% alcohol 30 s with 5% NaClO 15 min was the best.The optimal axillary buds induction medium was MS+0.5 mg·L^-1 6-BA+0.1 mg·L^-1 NAA with the maximum multiple buds ratio and buds height.After 20 d,the multiple buds ratio were 2.22 and buds height were 3.3 cm.The optimal subculture medium was MS+1.5 mg·L^-1 6-BA and the propagation coefficient could reach 4.1.The optimal rooting medium was 1/2MS+0.2 mg·L^-1 6-BA+1.0 mg·L^-1 NAA+0.02% activated carbon,with 12 d for the average rooting day,100% rooting rate and 1.04 cm root length.(2)The cultural method of the explants with single bud,was significantly (P＜0.05) higher than the cultural method of the single bud in the inductivity and average number of bulbils,which the inductivity,average number and size were 88.9%,1.50 and 0.38 cm×0.54 cm,respectively.1%~3% sucrose concentration was helpful to inducing the bulbils with had regular shape and the colour of the plantlets leaves were dark green.The optimal bulbils germination medium was MS+1.5 mg·L^-1 6-BA+0.2 mg·L^-1 NAA.The bulbils germinated at 18~22 d and inductivity reached 83.3% at 30 d.These results had a great potential application value for test-tube plantlet factory production of D.opposita Thunb.