A novel synthetic cry2A* gene was introduced into the elite indica rice restorer line Minghui 63 by Agrobacterium-mediated transformation. A total of 102 independent transformants were obtained. Among them, 71 transformants were positive cry2A* plants according to PCR analysis. Four highly insect-resistant lines with single-copy insertion (designated as 2A-1, 2A-2, 2A-3, and 2A-4) were selected based on field assessment and Southern blot analysis in the T(1) generation. All four transgenic lines showed Mendelian segregation by seed germination on 1/2 MS medium containing Basta. Homozygous transgenic plants were selected according to germination ratio (100%) in the T2 generation. Cry2A* protein concentrations were determined in homozygous transgenic lines, their derived hybrids, and their backcross offspring. The Cry2A* protein concentrations of four homozygous transgenic lines ranged from 9.65 to 12.11 microg/g of leaf fresh weight. There was little variation in the hybrids and backcross offspring. Insect bioassays were conducted in both the laboratory and field. All four transgenic lines were significantly resistant to lepidopteran rice pests. These cry2A* transgenic lines can be used to produce insect-resistant hybrids and serve as a resistant source for the development of two-toxin Bt rice.