The Arabidopsis T87 cell line has been widely used in both basic and biotechnological plant sciences. Agrobacterium-mediated transformation of this cell line was reported to be highly efficient when precultured in Gamborg's B5 medium for a few days. However, because we could not obtain the expected efficiency in our laboratory, we further examined the preculture conditions of Arabidopsis T87 cells in the Agrobacterium-mediated transformation. As a result, we found that preculture in an excess amount of Murashige and Skoog (MS) macronutrients before cultivation in the B5 medium enhanced the transformation efficiency up to 100-fold, based on the transformed callus number on selective gellan gum plates. In this study, transformants were labeled with green fluorescent protein (GFP), and we found multiple fluorescent spots on individual transgenic calli. Therefore, the actual number of transgenic clones seems much more than that of transgenic calli. In our MS macronutrient-rich culture condition, T87 cells tended to aggregate and formed bigger cell clumps, a change that might be related to the enhancement of transformation efficiency. Based on these results, we report an improved protocol of Agrobacterium-mediated transformation of Arabidopsis T87 cells with high efficiency.