Abstract: Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe (Aloe barbadensis Miller) by genetic engineering was seldom reported previously. In this study, regeneration and transformation conditions, including explant selection and surface sterilization, use of different Agrobacterium strains, and co-culture processing, are optimized. The use of 20.0% sodium hypochloride (25 min) for sterilization was less detrimental to the health of explant than 0.1% mercuric chloride (10 min). Regeneration frequency from stems was much higher than that from leaves or sheaths. Explants were infected by Agrobacterium (30 min) in liquid co-cultural medium, and this was followed by three days co-culture on sterile filter papers with light for 10 h per day at 24°C. Histochemical data demonstrated that the transient expression of GUS gene in the stem explants of aloe infected with Agrobacterium strains EHA105 and C58C1 was 80.0% and 30.0%, respectively, suggesting the higher sensitivity of the explants to EHA105 than to C58C1. Infected tissues were selected using G418 (10.0–25.0 mg/L) to generate transformants. Sixty-seven G418 resistant plantlets were generated from the infected explants. Southern blotting, PCR, and ELISA analyses indicated that the alien gene were successfully transferred into aloe and was expressed in the transgenic plants. This newly established transformation system could be used for the genetic improvement of aloe.