The crosstalk between cells and their microenvironment enables cellular adaptation to external mechanical cues through the remodeling of cytoskeletal structures and cell-matrix adhesions to ensure normal cell function. This study investigates the relationship between the cytoskeletal tension and integrin α5β1 adhesion strength to the matrix (i.e. fibronectin) in the context of RhoA-Src crosstalk. Integration of atomic force microscopy (AFM) with total internal reflection fluorescence and spinning-disk confocal microscopy enabled acquisition of complementary structural and functional measurements on live vascular smooth muscle cells expressing RhoA and c-Src variants (wild-type, dominant negative, constitutively active). Single ligand-receptor interaction measurements performed with AFM probes functionalized with fibronectin showed that RhoA and c-Src activation have different effects on cytoskeletal tension development, inducing two distinct force-stiffness functional regimes for α5β1-integrin binding to fibronectin. Moreover, fluorescence measurements showed that c-Src activation had a modest effect on actin morphology, while RhoA significantly modulated stress fiber formation. In addition, c-Src was associated with regulation of myosin light chain (MLC) phosphorylation, suggesting a c-Src-dependent modulation of RhoA pathway through activation of downstream effectors. Therefore, c-Src may be a possible component of cytoskeletal tension regulation through MLC activation. Our findings suggest that Src and RhoA coordinate a regulatory network that determines cytoskeletal tension through activation of actomyosin contractility. In turn, the cytoskeletal tension state modulates integrin α5β1-fibronectin adhesion force.