Establishment of efficient genome editing tools is essential for fundamental research, genetic engineering, and gene therapy. Successful construction and application of transcription activator-like effector nucleases (TALENs) in several organisms herald an exciting new era for genome editing. We describe the production of two active TALENs and their successful application in the targeted mutagenesis of silkworm, Bombyx mori, whose genetic manipulation methods are parallel to those of Drosophila and other insects. We will also show that the simultaneous expression of two pairs of TALENs generates heritable large chromosomal deletion. Our results demonstrate that (i) TALENs can be used in silkworm and (ii) heritable large chromosomal deletions can be induced by two pairs of TALENs in whole organisms. The generation and the high frequency of TALENs-induced targeted mutagenesis in silkworm will promote the genetic modification of silkworm and other insect species.