We report, exogenous addition of ceramide significantly increases 12-hydroxyeicosatetraenoic acid [12-(S)-HETE] levels, in a dose-dependent manner. 12-(S)-HETE levels, in 20, 30 and 40 μM ceramide exposed rat primary hippocampal cell cultures containing predominantly astrocytes and few neurons and other glial cells (the cultured hippocampal cells were predominantly astrocytes amounting to over 99% of total cells with few neurons and other glial cells) amounted to 207, 260 and 408% of the controls, respectively. However, dihydroceramide, an inactive analog of ceramide did not alter the levels of 12-(S)-HETE. Ceramide also increased the mRNA and protein expression, and activity of 12-lipoxygease (12-LOX) needed for the synthesis of 12(S)-HETE. These results indicate a possible link between ceramide and 12-LOX pathway. However, ceramide did not alter expression of 5-lipoxygenase (5-LOX), another member of the lipoxygenase family. However, ceramide upregulated expression of cytosolic phospholipase-A2 (cPLA2) and cyclooxygenase-2 (COX-2). Further, ceramide caused a significant increase in the levels of reactive oxygen species (ROS). Ceramide-mediated generation of ROS was inhibited by baicalien but not by indomethacin. In addition, ceramide treated cells exhibited increased mRNA expression of DNA damage induced transcript3 (Ddit3). This report which demonstrate induction of pro-carcinogenic 12-LOX pathway by an anticancer ceramide, may be relevant to cancer biologists studying drug resistant tumors and devising potent anticancer therapeutic strategies to treat drug resistant tumors. These results indicate possibility of 12-LOX involvement in ceramide-mediated generation of ROS and cellular oxidative stress. Induction of 12-LOX pathway by ceramide may have implications in understanding pathophysiology of neurodegenerative diseases involving ROS generation and inflammation.