Heat shock protein 27 (HSP27) modulates actin-dependent cell functions in several systems. We hypothesized that HSP27 modulates wound contraction. Stably transfected fibroblast cell lines that overexpress HSP27 (SS12) or underexpress HSP27 (AS10) were established, and cell behaviors related to wound contraction were examined. First, fibroblast-populated collagen lattice (FPCL) contraction was examined because it has been studied as a wound-healing model. In floating FPCL contraction assays, SS12 cells caused increased contraction, whereas AS10 cells caused reduced contraction. Because floating matrix contraction is thought to be mediated by the tractional force of the cells, cell behaviors related to tractional force were examined. In collagen matrix, SS12 cells elongated faster and to a greater extent and contained longer stress fibers than control cells, whereas AS10 cells were slower to elongate than control cells. SS12 cells attached to the dishes more efficiently than the control, whereas AS10 cells attached less efficiently. Migration of SS12 cells on collagen-coated dishes was also enhanced, although AS10 cells did not differ from the control cells. In summary, HSP27 regulates fibroblast adhesion, elongation, and migration and the contraction of the floating matrix in a manner dependent on the level of its expression.