Mechanical forces affect biological systems in their natural environment in a widespread manner. Mechanical stress may either stimulate cells or even induce pathological processes. Cells sensing mechanical stress usually respond to such stressors with proliferation or differentiation. Hence, for in vitro studies, the ability to impose a controlled mechanical stress on cells combined with appropriate analytical tools providing an immediate answer is essential to understand such fundamental processes. Here, we present a novel uniaxial motorized cell stretching device that has been integrated into a combined fluorescence microscope (FM)-atomic force microscope (AFM) system, thereby enabling high-resolution topographic and fluorescent live cell imaging. This unique tool allows the investigation of mechanotransduction processes, as the cells may be exposed to deliberately controlled mechanical stress while simultaneously facilitating fluorescence imaging and AFM studies. The developed stretching device allows applying reproducible uniaxial strain from physiologically relevant to hyperphysiological levels to cultured cells grown on elastic polydimethylsiloxane (PDMS) membranes. Exemplarily, stretching experiments are shown for transfected squamous cell carcinoma cells (SCC-25) expressing fluorescent labeled cytokeratin, whereby fluorescence imaging and simultaneously performed AFM measurements reveal the cytokeratin (CSK) network. Topographical changes and mechanical characteristics such as elasticity changes were determined via AFM while the cells were exposed to mechanical stress. By applying a cell deformation of approx. 20%, changes in the Young's modulus of the cytoskeletal network due to stretching of the cells were observed. Consequently, integrating a stretching device into the combined atomic force-fluorescence microscope provides a unique tool for dynamically analyzing structural remodeling and mechanical properties in mechanically stressed cells.