Bacillus thuringiensis is a Gram-positive, ubiquitous, spore-forming bacterium that produces large amounts of proteins that crystallize inside the cell during the sporulation stage known as B. thuriniensis delta-endotoxins or Cry proteins. The insecticidal Cry proteins produced by B. thuringiensis have provided a particular, secure and effective tool for the control of a wide diversity of insect pests around the world for over 70 years. The Cry proteins are lethal to insect larvae in the orders Lepidoptera, Diptera and Coleoptera. More recently, isolates have been identified with activity against the organisms in the orders Hymenoptera, Homoptera, Orthoptera and Mallophaga and also nematodes, mites, lice and protozoa. Furthermore, in the global biopesticide market B. thuringiensis represents nearly US$600 million/year. At least 900 different Cry toxin sequences have been found and classified into 73 family groups (by 2014). Biotechnology and genetic manipulation of cry genes present in B. thuringiensis can potentially improve the efficacy and cost-effectiveness of B. thuringiensis-based commercial products. The combination of genes from different B. thuringiensis strains to enhance their activity, extend their host range and improve the spectrum of insecticidal activity has been achieved with recombinant technologies. These genetically modified B. thuringiensis products are currently commercially available. It has been recently established that Cry hybrid proteins of B. thuringiensis, gained by domain swapping, resulted in enhanced toxicities when compared with wild-type proteins. Nowadays, B. thuringiensis insecticidal genes have been included in several of the most important crop plants where they provide a model for biotechnology in agriculture. The B. thuringiensis transgenic crop has received more attention in cases where cry genes have been brought together by a mixture of mutagenesis and oligonucleotide synthesis to produce synthetic genes. In this chapter, manipulation of Cry proteins from the soil bacterium B. thuringiensis and its biotechnological applications are described. The future prospects are also discussed.