Background. Cell surface mechanics is able to physically and biomechanically affect cell shape and motility, vesicle trafficking and actin dynamics. The biophysical properties of cell surface are strongly influenced by cytoskeletal elements. In mammals, tissue-specific expression of six actin isoforms is thought to confer differential biomechanical properties. However, the relative contribution of actin isoforms to cell surface properties is not well understood. Here, we sought to investigate whether and how the composition of endogenous actin isoforms directly affects the biomechanical features of cell surface and cellular behavior. Methods: We used fibroblasts isolated from wild type (WT), heterozygous (HET) and from knockout (KO) mouse embryos where both beta-actin alleles are not functional. We applied a combination of genome-wide analysis and biophysical methods such as RNA-seq and atomic force microscopy. Results: We found that endogenous beta-actin levels are essential in controlling cell surface stiffness and pull-off force, which was not compensated by the up-regulation of other actin isoforms. The variations of surface biophysical features and actin contents were associated with distinct cell behaviors in 2D and 3D WT, HET and KO cell cultures. Since beta-actin in WT cells and smooth muscle alpha-actin up-regulated in KO cells showed different organization patterns, our data support the differential localization and organization as a mechanism to regulate the biophysical properties of cell surface by actin isoforms. Conclusions: We propose that variations in actin isoforms composition impact on the biophysical features of cell surface and cause the changes in cell behavior.