The microtubule-dependent motor protein Eg5 plays a critical role in spindle assembly and maintenance in mitosis. Herein we show that the suppression of Eg5 by a specific inhibitor arrested mitosis, induced apoptosis, and up-regulated Hsp70 in human multiple myeloma cells. Mechanistically, Hsp70 induction occurred at the transcriptional level via a cis-regulatory DNA element in Hsp70 promoter and was mediated by the phosphatidylinositol 3-kinase/Akt pathway. Eg5 inhibitor-mediated Hsp70 up-regulation is cytoprotective because blocking Hsp70 induction directly by antisense or small interfering RNA or indirectly by inhibiting the phosphatidylinositol 3-kinase/Akt pathway significantly increased Eg5 inhibitor-induced apoptosis. Furthermore, a farnesyltransferase inhibitor interacted synergistically with the Eg5 inhibitor in inducing apoptosis through disrupting the Akt/Hsp70 signaling axis. These findings provide the first evidence for Eg5 inhibitor activity in hematologic malignancy and identify Hsp70 up-regulation as a critical mechanism responsible for modulating myeloma cell sensitivity to Eg5 inhibitors. In addition, these findings suggest that a combination of Eg5 inhibitors with agents abrogating Hsp70 induction would be useful for myeloma therapy in the clinic.