Among the many processes regulating cell death, ceramide signaling is a vital component. We previously determined that acid ceramidase (AC) is upregulated in 60% of primary prostate cancer (PCa) tissues, suggesting that AC may play a role in tumor development. In order to determine the significance of AC elevation, stable clones of DU145 cells with AC over-expression (AC-EGFP) were generated. Compared to controls (EGFP), AC-EGFP cells exhibited enhanced cell proliferation and migration. Subcutaneous injection of AC-EGFP cells into Nu/Nu mice resulted in larger tumor volumes compared to EGFP controls. Moreover, using the MTS viability assay, AC-EGFP cells were more resistant to cell death induced by doxorubicin, cisplatin, etoposide, gemcitabine or C6-ceramide. Conversely, knock down of AC using siRNA, sensitized AC-EGFP cells to these drugs. In addition, mass spectroscopic analysis of sphingolipids indicated that long chain ceramide levels were decreased in AC-EGFP cells treated with either doxorubicin or etoposide. In conclusion, this study implicates AC as a critical regulator of PCa progression by affecting not only tumor cell proliferation and migration but also responses to drug therapy, suggesting AC as a potential therapeutic target in advanced PCa.