Hyperthermia adversely affects cell structure and function, but also induces adaptive responses that allow cells to tolerate these stressful conditions. For example, heat-induced expression of the molecular chaperone protein HSP70 can prevent stress-induced cell death by inhibiting signaling pathways that lead to apoptosis. In this study, we used high-resolution two-dimensional gel electrophoresis and phosphoprotein staining to identify signaling pathways that are altered by hyperthermia and modulated by HSP70 expression. We found that in heat-shocked cells, the actin-severing protein cofilin acquires inhibitory Ser3 phosphorylation, which is associated with an inhibition of chemokine-stimulated cell migration. Cofilin phosphorylation appeared to occur as a result of the heat-induced insolubilization of the cofilin phosphatase slingshot (SSH1-L). Overexpression of HSP70 reduced the extent of SSH1-L insolubilization and accelerated its resolubilization when cells were returned to 37°C after exposure to hyperthermia, resulting in a more rapid dephosphorylation of cofilin. Cells overexpressing HSP70 also had an increased ability to undergo chemotaxis following exposure to hyperthermia. These results identify a critical heat-sensitive target controlling cell migration that is regulated by HSP70 and point to a role for HSP70 in immune cell functions that depend upon the proper control of actin dynamics.