In this study, a highly efficient plant regeneration and Agrobacterium-mediated genetic transformation protocols of lisianthus ‘Micky Rose’ were established. Immature and mature internodal explants were incubated with Agrobacterium tumefaciens strain LBA4404 containing a binary vector pBAL2 and carrying the reporter gene β-glucuronidase intron (GUS-INT), and the marker gene neomycin phosphotransferase (NPTII). Factors affecting transformation efficiency, such as effect of pre-culture, Agrobacterium concentration, acetosyringone, infection, and co-cultivation time of Agrobacterium, were investigated. After co-cultivation, explants were transferred onto MS medium supplemented with 1.0 µM α-naphthalene acetic acid (NAA) and 1.5 µM thidiazuron (TDZ), 100 mg l^-1 kanamycin, and 300 mg l^-1 carbenicillin for callus induction. Regeneration of adventitious shoots from callus was achieved on Murashige and Skoog (MS) medium containing 3.0 µM TDZ, 0.5 µM NAA, 100 mg l^-1 kanamycin, and 300 mg l^-1 carbenicillin. Immature internodal explants produced more shoots (30 shoots) than mature internodal explants. The transgenic elongated shoots were rooted (91.3%) on MS medium supplemented with 3.0 μM indole-3-acetic acid (IAA) and 100 mg l^-1 kanamycin. Three days pre-cultivation, Agrobacterium density, infection time for 30 min, acetosyringone (300 μM), and four days of co-cultivation proved to be critical factors for greatly improving the transformation efficiency. Molecular studies of transgenic plants and their offspring confirmed the presence of the nptII gene. Transgenic plants were acclimatized (90%) in the greenhouse and transgenic efficiency of 21% was evaluated.