Many forms of cellular stress cause an elevation of endogenous ceramide levels leading to growth arrest or apoptosis. Ceramidases (CDase) play a critical role in regulating apoptosis by hydrolyzing ceramide into sphingosine, a precursor for promitogenic sphingosine-1-phosphate. Growth factor induction of neutral CDase (nCDase) has been shown to have a cytoprotective effect against cytokine-induced increases in ceramide levels. To further define the physiological regulation of nCDase, we identified a 200 bp promoter region and demonstrated that serum activated this proximal promoter, which correlated with a serum-induced increase in human nCDase mRNA expression. Computational analysis revealed a putative cis-element for AP-1, a transcription factor activated by serum. Electrophoretic mobility shift assays demonstrated that the identified transcriptional response element binds to AP-1 transcription factors. RNA interference-mediated knockdown of the AP-1 subunit, c-Jun, inhibited the activity of the human nCDase proximal promoter, whereas, c-Jun overexpression increased promoter activity, which directly correlated with human nCDase mRNA transcription, decreased ceramide mass, and protection against caspase 3/7-dependent apoptosis. Taken together, our findings suggest that c-Jun/AP-1 signaling may, in part, regulate serum-induced human nCDase gene transcription.