NF-κB signalling is one of the main cell survival pathways that attenuate the anticancer efficacy of therapeutic drugs. Previous studies demonstrated that the histone deacetylase (HDAC) inhibitor induces apoptosis in some malignancies through multiple mechanisms including up-regulation of death receptors, disruption of Hsp90 function and generation of reactive oxygen species (ROS). However, HDAC inhibitor also induces a cell survival signal through NF-κB activation. In this report, we found that romidepsin, a class I HDAC inhibitor, induces NF-κB activation in A549 non-small-cell lung cancer (NSCLC) cells. We also found that inhibition of A549 cells with bortezomib (proteasome inhibitor) has blocked IκB degradation that leads to the loss of NF-κB activation and translocation which enhanced the romidepsin induced mitochondrial injury and sensitizes NSCLC cells to apoptosis. Romidepsin significantly enhances NF-κB reporter gene transcription and these effects were inhibited by bortezomib as determined by reporter gene assay. Consistently, the combined exposure of romidepsin and bortezomib reversed the effects on IκB degradation as evident with IL-8, p50 and p65 (NF-κB) expression. Apoptosis was markedly sensitized with greater ROS generation and more cell death in A549 cell lines. These events are most closely related in that bortezomib prevents the romidepsin mediated RelA acetylation and NF-κB activation, resulting in caspase activation. A strategy of blocking NF-κB activation to enhance HDAC inhibitor activity warrants further attention in NSCLC cells.