Solanum trilobatum L. is one of the most valued medicinal plants of Indian Ayurveda and Siddha medicine. An efficient protocol for direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation using in vitro derived leaf explants of S. trilobatum was established for the first time. High frequency direct shoot regeneration (89%) was achieved in MS medium containing 2 mg/L 6-benzylaminopurine and 0.25 mg/L indole-3-acetic acid. The Agrobacterium strain EHA 105 harboring the binary vector pCAMBIA 1301, which contained hygromycin phosphotransferase (hpt) as a selectable marker gene and β-glucuronidase (gus) as a reporter gene, was used for the transformation. The factors that influence the transformation frequency were optimized as follows: age of explants (40 days old), acetosyringone concentration (150 µM), duration of preculture (2 days), bacterial optical density (O.D. 0.4), Tween 20 concentration (0.01%), infection time (15 min), pH of cocultivation medium (5.4), and cocultivation duration (3 days). The putative transformants were screened on selection medium containing 15 mg/L hygromycin. The transgenic plants were confirmed by histochemical gus analysis, PCR, and RT-PCR analysis. A stable transformation efficiency of 64% was achieved under optimized transformation conditions. Thus, the protocol can be used for genetic improvement of this plant in the future.