Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases and a correlated Programmed Cell Death (PCD) in tobacco BY-2 cells. In this study, in order to get deeper insight into the LCB signaling pathway leading to cell death, the putative role of Reactive Oxygen Species (ROS) has been investigated. We show that DHS triggers a rapid dose-dependent production of H₂O₂ that is blocked by diphenyleniodonium (DPI), indicating the involvement of NADPH oxidase(s) in the process. In addition, while DPI does not block DHS-induced calcium increases, the ROS production is inhibited by the broad spectrum calcium channel blocker lanthanum (La³+). Therefore, ROS production occurs downstream of DHS-induced Ca²+ transients. Interestingly, DHS activates expression of defense-related genes that is inhibited by both La³+ and DPI. Since DPI does not prevent DHS-induced cell death, these results strongly indicate that DHS-induced H₂O₂ production is not implicated in PCD mechanisms but rather would be associated to basal cell defense mechanisms.