Cotton (Gossypium hirsutum L.) is an important fiber crop in the world. Abiotic and abiotic stresses are the main challenges of yield reduction in cotton. Traditionally, four to six times of spraying per growing season of environmentally harmful chemical insecticides is used for pest control. There are various methods for producing transgenic cotton, among which the Agrobacterium tumefaciens method is widely used. In order to produce transgenic cotton tolerance to stresses, explants are transformed with Agrobacterium tumefaciens. Also, selectable marker gene is used as a marker. Inoculated tissue sections are placed onto co-cultivation medium. Transformed tissues are selected on culture medium containing suitable antibiotic. Plantlets are subsequently regenerated from putative transgenic explants. In this chapter, the steps of genetic transformation of cotton by Agrobacterium method using embryogenic calli have been completely explained.