Lilium browniivar.viridulum, commonly called Longya lily, is a well-known flower and vegetable plant in China that has poor tolerance toBotrytisfungal disease. The molecularimprovement has mainly been restricted to an efficient regeneration and transformation system. In this study, the highly efficient regeneration of Longya lily was established through the optimization of embryogenic callus, adventitious shoot and rooting induction. The major factors influencing transformation (antibiotics,Agrobacteriumconcentration, infection time, suspension solution and coculture medium) were examined. The expression responses of PR promoters (ZmPR4andBjCHI1) toB. cinereawere assessed in transgenic calli. The results showed that Murashige and Skoog (MS) medium with 1.0 mg·L−1picloram (PIC) and 0.2 mg·L−11-naphthaleneacetic acid (NAA) under light conditions and MS with 0.5 mg·L−16-benzylaminopurine (6-BA) and 1.0 mg·L−1NAA under darkness were optimal for embryogenic callus induction (64.67% rate) and proliferation (3.96 coefficient). Callus inoculation into MS containing 2.0 mg·L−1thidiazuron (TDZ), 0.4 mg·L−1NAA, 1.0 mg·L−1TDZ and 0.5 mg·L−1NAA led to shooting induction (92.22 of rate) and proliferation (3.28 of coefficient) promotion, respectively. The rooting rate reached 99.00% on MS with 0.3 mg·L−1NAA. Moreover, a transformation rate of 65.56% was achieved by soaking the callus inAgrobacteriumat an OD600of 0.4 for 10 min in modified MS without NH4NO3as the suspension solution and coculture medium before selecting 75 mg·L−1hygromycin and 300 mg·L−1cefotaxime. Only theBjCHI1promoter was obviously expressed in transgenic calli. These results could facilitate the generation of Longya lily transgenic plants with improvedB. cinerearesistance.