Understanding the structural changes and property alterations at the nanoscale and microscopic levels is critical to clarifying the deformation behavior and mechanical properties of polymer materials. Especially, in latex films composed of polymer nanoparticles, it is widely accepted that the remaining interfaces between microparticles in the film affect their brittleness. However, detailed information on nanoscale changes of latex films during deformation remains unclear due to technical difficulties in analyzing the microstructures under mechanical stress. In this study, we employed atomic force microscopy equipped with a uniaxial stretching device to visualize the surface structures of films composed of slightly cross-linked microparticles under elongation strain. The observations revealed that the latex film deforms in a nonaffine manner, which is attributed to the concurrent deformation of individual microparticles and the pull-out of interpenetration between them. Furthermore, by introducing a load−strain measurement mechanism to the stretching device, we compared the relationships between nanostructural changes, local property changes, and macroscopic deformation of microparticle-based films. The results suggest that loads are dominated by the deformation of microparticles and dissipate as the interpenetration of surface polymer chains between microparticles is pulled out.