We have previously shown that heat shock protein (Hsp) 27 or its upstream activator p38 mitogen-activated protein kinase (MAPK) confers resistance to bortezomib and dexamethasone (Dex) in multiple myeloma (MM) cells. This study examined anti-MM activity of a novel p38 MAPK inhibitor, BIRB 796, alone and in combination with conventional and novel therapeutic agents. BIRB 796 blocked baseline and bortezomib-triggered upregulation of p38 MAPK and Hsp27 phosphorylation, thereby enhancing cytotoxicity and caspase activation. The Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) upregulated protein expression and phosphorylation of Hsp27; conversely, BIRB 796 inhibited this phosphorylation and enhanced 17-AAG-induced cytotoxicity. Importantly, BIRB 796 inhibited Hsp27 phosphorylation induced by 17-AAG plus bortezomib, thereby enhancing cytotoxicity. In bone marrow stromal cells (BMSC), BIRB 796 inhibited phosphorylation of p38 MAPK and secretion of interleukin-6 (IL-6) and vascular endothelial growth factor triggered by either tumour necrosis factor-α or tumour growth factor-β1. BIRB 796 also inhibited IL-6 secretion induced in BMSCs by adherence to MM cells, thereby inhibiting tumour cell proliferation. These studies therefore suggest that BIRB 796 overcomes drug-resistance in the BM microenvironment, providing the framework for clinical trials of a p38 MAPK inhibitor, alone and in combination with bortezomib, Hsp90 inhibitor, or Dex, to improve patient outcome in MM.