Bt crops are the most important of the commercialised transgenic plants which were developed to combat destructive pests. Colorado potato beetle (CPB) is considered as a dangerous threat to potato production. In this study transgenic potato plants expressing cry3A gene were generated using Agrobacterium mediated transformation to combat the pest damage. Polymerase chain reaction (PCR) analysis confirmed the presence of transgenes in 40 out of 110 putative transgenic lines. One to three copies of cry3A gene were identified in selected transgenic lines using southern blot analysis. Moreover, immunostrip test confirmed the expression of Cry3A protein in the leaves of transgenic potato plants. Bioassay analysis with the first instar of CPB larvae indicated 100% mortality, significantly lower leaf damage and less insect biomass accumulation for transgenic lines (PC3-3, PC3-4, PC3-5, PC3-8 and PC3-13) in comparison to control plants. The results indicated that Cry3A protein expression could efficaciously increase the resistance to CPB in transgenic potato cv. Agria which is recommended for controlling CPB damage and to prevent its spread in potato cultivation regions. Moreover, these transgenic lines can be used as cultivar or germplasm to breed new cultivars.