Conventional breeding methods have not been very successful in producing pest-resistant genotypes of pigeonpea, due to the limited genetic variation in cultivated germplasm. We have developed an efficient method to produce transgenic plants of pigeonpea by incorporating the cry1Ab gene of Bacillus thuringiensis through Agrobacterium tumefaciens-mediated genetic transformation. The novel tissue culture protocol is based on the direct regeneration of adventitious shoot buds in the axillary bud region of in vitro germinating seedlings by suppressing the axillary and primary shoot buds on a medium containing a high concentration of N⁶-benzyladenine (22.0 μM). The tissue with potential to produce adventitious shoot buds can be explanted and used for co-cultivation with A. tumefaciens carrying the synthetic cry1Ab on a binary vector and driven by a CaMV 35S promoter. Following this protocol, over 75 independently transformed transgenic events of pigeonpea were produced and advanced to T2 generation. Amongst the recovered primary putative transformation events, 60% showed positive gene integration based on initial polymerase chain reaction (PCR) screening. PCR analysis of the progenies from independent transformants followed gene inheritance in a Mendelian ratio and 65% of the transformants showed the presence of single-copy inserts of the introduced genes. Reverse transcription–polymerase chain reaction analysis showed that the transcripts of the introduced genes were normally transcribed and resulted in the expression of Cry1Ab protein in the tested T2 generation plants. Interestingly, the content of Cry1Ab protein as a percent of total soluble protein varied in different tissues of the whole plant, showing the highest expression in flowers (0.1%) and least in the leaves (0.025%) as estimated by enzyme-linked immunosorbent assay. The transgenic plants produced in this study offer immense potential for the improvement of this important legume of the semi-arid tropics for resistance to insect pests.