The question of specificity and the elucidation of the exact molecular mechanism of action of post-transcriptional gene silencing agents are two major challenges for their establishment as therapeutics. A proteomic off-target effect study (2-DE with MS) in combination with DIGE comparing the phosphorothioate antisense oligonucleotide oblimersen (Genasense, G3139) to a Bcl-2-targeting siRNA-sequence on human melanoma cells showed that additional off-target effects contribute to the apoptotic effect of oblimersen. When both oligonucleotides were transfected with lipofectamine 2000, only oblimersen increased apoptosis as determined by annexin staining and caspase activity measurement. In contrast to the highly specific siRNA, the expression level of a number of proteins was found to be altered after oblimersen treatment. Several proteins linked to apoptosis and stress response, among those galectin-1, cofilin-1, GRP78, HSP60, nucleophosmin, and peroxiredoxins, were identified and found to be down-regulated after oblimersen treatment. A down-regulation of enolase-1 and three other glycolytic enzymes indicates a reversion of the cancer-related Warburg effect. The observed effects may be caused by a phosphorothioate mediated blockage of the mitochondrial voltage dependent anion channel (VDAC).