Recent evidence demonstrates that the anticancer activity of betulinic acid (BetA) can be markedly increased by combination protocols, for example with chemotherapy, ionizing radiation or TRAIL. Since nuclear factor-kappaB (NF-kappaB), a key regulator of stress-induced transcriptional activation, has been implicated in mediating apoptosis resistance, we investigated the role of NF-kappaB in BetA-induced apoptosis. Here, we provide for the first time evidence that BetA activates NF-kappaB in a variety of tumor cell lines. NF-kappaB DNA-binding complexes induced by BetA consisted of p50 and p65 subunits. Nuclear translocation of p65 was also confirmed by immunofluorescence microscopy. BetA-induced NF-kappaB activation involved increased IKK activity and phosphorylation of IkappaB-alpha at serine 32/36 followed by degradation of IkappaB-alpha. Reporter assays revealed that NF-kappaB activated by BetA is transcriptionally active. Interestingly, inhibition of BetA-induced NF-kappaB activation by different chemical inhibitors (proteasome inhibitor, antioxidant, IKK inhibitor) attenuated BetA-induced apoptosis. Importantly, specific NF-kappaB inhibition by transient or stable expression of IkappaB-alpha super-repressor inhibited BetA-induced apoptosis in SH-EP neuroblastoma cells, while transient expression of IkappaB-alpha super-repressor had no influence on BetA-induced apoptosis in two other cell lines. Thus, our findings that activation of NF-kappaB by BetA promotes BetA-induced apoptosis in a cell type-specific fashion indicate that NF-kappaB inhibitors in combination with BetA would have no therapeutic benefit or could even be contraproductive in certain tumors, which has important implications for the design of BetA-based combination protocols.