Cell survival reflects a balance between death and protective pathways. Because sphingolipids have emerged as putative death signals and because transcription factor‐kappa B (NF‐κB) activates a survival pathway, we analyzed the role of C2‐ceramide (C2) and GD3 ganglioside (GD3) on the generation of reactive oxygen species (ROS), NF‐ΚB activation, and survival of rat hepatocytes or HepG2 cells. Although both C2 and GD3 generated similar dose‐dependent levels of ROS derived from mitochondria, hepatocytes displayed a selective sensitivity to GD3 treatment. Consistent with this finding, C2 and GD3 differed in the activation of NF‐ΚB as C2, unlike GD3, and enhanced the DNA binding of NF‐ΚB despite the fact that both sphingolipids signaled the degradation of IΚB‐Α. Glucosylceramide (GluCer), lactosylceramide (LactCer), and ganglioside GM1 (GM1) mimicked the repressing effect of GD3 on NF‐ΚB activation, which suggests the presence of common structural features among these glycosphingolipids. Competent DNA binding NF‐ΚB complexes were observed predominantly in the cytosol of GD3‐stimulated cells, which paralleled the absence of NF‐ΚB p65 in the nuclei and indicated that GD3 blocks the nuclear translocation of NF‐ΚB complexes. Pretreatment of hepatocytes with a sublethal dose of GD3 blocked the activation of NF‐ΚB and subsequent ΚB‐dependent gene expression induced by tumor necrosis factor α (TNF‐α), which sensitized hepatocytes to TNF‐α‐induced apoptosis. Thus, gangliosides are efficient death effectors by a dual mechanism that involves mitochondrial recruitment and suppression of the NF‐κB‐dependent survival pathway, which may be of potential therapeutic use in conditions aimed to control apoptosis resistance.