AbstractGentiana rhodanthais a perennial herb of the genusGentiana(Tourn.) L. This study was novel in establishing a regeneration system ofG. rhodanthausing young leaves as explants on the Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs). The roots, stems, and leaves ofG. rhodanthawere used as explants. The effects of the optimal explant disinfection method, type of explant used, concentrations of PGRs added to the culture media on tissue culture, and rapid propagation ofG. rhodanthawere studied. The results showed that the optimal disinfection method for stems and roots consisted of disinfection using 75% ethanol for 50 s, followed by 4% sodium hypochlorite (NaClO) for 10 min. The optimal disinfection technique for leaves consisted of disinfection using 75% ethanol for 50 s, followed by 4% NaClO for 8 min. Root explant was the most suitable for inducing the callus ofG. rhodanthaon the MS medium supplemented with different PGRs. The optimal conditions for callus induction included 1.0 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L α-naphthalene acetic acid (NAA). The callus induction rate using the root explant reached 94.28%. MS supplemented with 2.0 mg/L 6-BA and 0.1 mg/L NAA was the optimal medium for inducing adventitious shoots from the callus ofG. rhodantha. The best medium for propagation and plantlets strengthening was MS supplemented with 0.8 mg/L 6-BA and 0.3 mg/L NAA, and the propagation index was 8.62. MS supplemented with 0.3 mg/L 3-indolebutyric acid was the best culture medium for inducing the rooting of adventitious buds, with the maximum rooting rate reaching up to 100%.