Populus species are important resources for industry and in scientific study on biological and agricultural systems. Our objective was to enhance the frequency of plant regeneration in Himalayan poplar (Populus ciliata wall. ex Royle). Agrobacterium-mediated plant genetic transformation requires a two step process for its success, selection and regeneration of transformed tissues and the elimination of the transformation vector Agrobacterium. Antibiotics used for suppressing Agrobacterium in plant transformation might have negative effects on plant tissues and regeneration. The effects of antibiotics on growth suppression of Agrobacterium and plant regeneration were investigated for enhancing Agrobacteriummediated transformation. This study uses cefotaxime with combination of kanamycin to eliminate Agrobacterium tumefaciens LBA4404 and selection of transgenic plant. First the effect of antibiotic cefotaxime was evaluated during in vitro shoot morphogenesis in Populus ciliata L. Later on effect of different concentrations of cefotaxime and kanamycin (50 mg/l) were studied on the growth of agrobacterial cells and regeneration potential of leaf and petiole tissues after co-cultivation. The maximum per cent (75%) and (78%) shoot regeneration were obtained on MS regeneration medium with 300mg/l cefotaxime in leaf and petiole explants, respectively. In leaf and petiole explants the growth of agrobacterial cells were controlled at concentration of 400 mg/l cefotaxime and maximum per cent shoot regeneration 33.00% and 48.00% was obtained on MS medium supplemented with 400 mg/l cefotaxime, respectively. A protocol for high frequency transformation of insect resistance gene in Himalayan poplar have been standardized.