Abstract: Rice has been transformed via Agrobacterium transfection with use of mature embryo-derived calli, but the transformation efficiency has remained relatively low in general. In this study, we optimized different factors affecting transformation and established a highly efficient transformatiom system mediated by Agrobacterium using mature embryo-derived calli from two japonica cultivars (Zhonghua 10, 11). The transformation was performed with A. tumefaciens strain EHA105 harboring the plasmid pUN1301/OsRAA1, GUS used as the reporter gene and HPT as the selectable marker gene. First, we established a high-frequency regeneration system for rice, defined NBD2 (NB+2 mg·L-1 2,4-D) as callus induction medium, NBD0.5 as subculture medium, and RE1 (MS+1 mg·L-1 6-BA+0.25 mg·L-1 NAA+0.5 mg·L-1 KT+0.2 mg·L-1 ZT) and RE2 (MS+1 mg·L-1 6-BA+0.5 mg·L-1 NAA+0.5 mg·L-1KT+0.2 mg·L-1 ZT) as regeneration medium. Second, factors such as the selection of explants, the pre-culture medium and the subculture times were shows to affect the efficiency of T-DNA delivery. The transformation efficiency using the optimized method was increased to 70%, which was measured either by counting the number of hygromycin-resistant calli or the number of transgenic plants.