Leptomycin B (LMB) is a Streptomyces metabolite that inhibits the chromosomal region maintenance (CRM)1-dependent nuclear export of proteins. It also induces apoptosis in several types of cancer cells, by a yet undefined mechanism. We used a functional proteomics approach to delineate the pathways and mediators involved in LMB-induced apoptosis in HeLa cells. Using two-dimensional gel electrophoresis, we searched for proteins accumulated in the nuclei of HeLa cells upon LMB treatment. Among such proteins we found prohibitin and Heat shock protein (Hsp)27, identified by peptide mass fingerprinting and mass spectrometry. Immunocytochemistry was carried out to confirm the LMB-induced nuclear accumulation of these two proteins in HeLa cells. Furthermore, we found that the cytoplasmic expression of Hsp27, but not prohibitin, partially inhibited LMB-induced apoptosis, indicating that nuclear sequestration of Hsp27 was, at least in part, involved in the apoptosis.