Agrobacterium tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency of coculture, selection cultivation, and plant regeneration. The binary vector pBI121 (containing a neomycin phosphotransferase II gene npt-II as a selection marker and a uidA gene as a reporter gene) was used to research transformation efficiency. After 48 h cocultivation, the number of β-glucuronidase (GUS)-positive calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse granule EC. It indicated that the efficiency of transient transformation was affected by EC morphology. Transient transformation efficiency also was improved by cocultivation on the medium by adding 50 mg/L acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density increased the production of kanamycin-resistant (Km-R) calli lines. From an original 0.3 g EC, an average of 20 Km-R calli lines were obtained from a selection dish, and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated on the differentiation medium with dehydration treatments, and the GUS-positive rate of regeneration plants was about 72.6%. Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase II gene and gus gene. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly with the low copy number, was integrated into the cotton genome.