Background: We previously reported that N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) treatment caused large increases of ceramide levels in neuroblastoma cell lines and induced cell death by a combination of apoptosis and necrosis through p53 (also known as TP53)-independent and caspase-independent pathways. Our goal was to determine if several molecules that inhibit enzymes involved in ceramide metabolism—l-threo-dihydrosphingosine (safingol), d,l-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), and tamoxifen—enhanced 4-HPR-mediated cytotoxicity and/or affected ceramide levels. Methods: Cellular lipids were quantified by radiolabeling and thin-layer chromatography. Cytotoxicity and cytotoxic synergy (expressed as combination index, where combination index <1 indicates synergy and >1 indicates antagonism) were measured in cultured cancer cell lines with the use of a fluorescence-based assay of cell viability employing digital imaging microscopy. Statistical tests were two-sided. Results: 4-HPR increased ceramide levels by de novo synthesis. Safingol (1–4 μM) was incorporated into a stereochemical variant of ceramide and synergized with a 3 : 1 molar ratio of 4-HPR (3–12 μM), to produce a 100-fold to 10 000-fold (2 to 4 logs) increase in cytotoxicity relative to 4-HPR alone in neuroblastoma (combination index <0.1), lung (combination index <0.1–0.2), melanoma (combination index <0.1–0.2), prostate (combination index <0.1–1.0), colon (combination index 0.1–0.3), breast (combination index = 0.1–0.5), and pancreas (combination index = 0.2) cell lines, including p53 mutant and alkylator-resistant cell lines. The 4-HPR and safingol combination was cytotoxic in low-oxygen conditions and was minimally toxic to normal fibroblasts and bone marrow myeloid progenitor cells. Addition of agents that retard ceramide glucosylation and/or acylation, such as PPMP or tamoxifen, to 4-HPR or to the combination of 4-HPR and safingol further increased cytotoxicity to tumor cells. Conclusions: Combinations of 4-HPR and modulators of ceramide metabolism may form the basis for a novel chemotherapy that is functional under hypoxic conditions (e.g., such as those within tumors) and is p53 independent and caspase independent.