<jats:title>Abstract</jats:title><jats:p>Development of chemoresistance in androgen‐refractory prostate cancer cells is partly due to constitutive activation of Rel/NF‐κB transcription factors that regulate several cell survival and anti‐apoptotic genes. In this study we examined whether betulinic acid (BetA), a pentacyclic triterpene from the bark of white birch, is effective in inhibiting NF‐κB expression in androgen‐refractory human prostate cancer cells exhibiting high constitutive NF‐κB expression. Treatment of PC‐3 cells with BetA inhibited DNA binding and reduced nuclear levels of the NF‐κB/p65. BetA‐mediated NF‐κB inhibition involved decreased IKK activity and phosphorylation of IκBα at serine 32/36 followed by its degradation. Reporter assays revealed that NF‐κB inhibition by BetA is transcriptionally active. These effects were found to correlate with a shift in Bax/Bcl‐2 ratio and cleavage of poly(ADP)ribose polymerase more towards apoptosis. BetA also inhibited TNFα‐induced activation of NF‐κB via the IκBα pathway, thereby sensitizing the cells to TNFα‐induced apoptosis. Our studies demonstrate that BetA effectively inhibits constitutive NF‐κB activation and supports the rationale for targeting NF‐κB through combination protocols with BetA in androgen‐refractory prostate cancer. © 2008 Wiley‐Liss, Inc.