We previously established an experimental system for efficient pathogenic signal induced-cell death using tobacco BY-2 cells and culture filtrate of a plant pathogenic bacterium, Erwinia carotovora. Using this experimental system, cytoskeletal and vacuolar changes during the process of filtrate-induced cell death were characterized in detail. However, the initial events induced by the filtrate were largely unknown. In this study, reactive oxygen species (ROS) production and endocytosis stimulation were examined in BY-2 cells treated with the filtrate of E. carotovora. Transient ROS production within 2 min was observed after the filtrate treatment. Treatment with the Ca2+ chelator BAPTA or the protein kinase inhibitor K252a significantly inhibited ROS production in a dose-dependent manner, suggesting that ROS production depends on Ca2+ influx or protein phosphorylation. In addition, internalization of the endocytic marker FM4-64 was promoted by filtrate treatment within 30 min, suggesting that the filtrate treatment stimulated endocytosis. These results showed that ROS production and subsequent endocytosis stimulation are the initial events induced by culture filtrate of E. carotovora in BY-2 cells.