Stiffening of the extracellular matrix (ECM) occurs after vascular injury and contributes to the injury-associated proliferation of vascular smooth muscle cells (SMCs). ECM stiffness also activates Rac-GTP, and SMC Rac1 deletion strongly reduces the proliferative response to injury in vivo. While these resultss strongly implicate Rac in the stiffness-dependent SMC proliferation, ECM stiffening and Rac also affect SMC differentiation, which, in itself, can influence cell stiffness and proliferation. Here, we used immunofluorescence analysis and pressure myography of mouse carotid arteries to interrogate the effect of in vivo Rac1 deletion on SMC differentiation and arterial stiffness. The results show that medial abundance of alpha-smooth muscle actin or smooth muscle-myosin heavy chain, markers of the SMC differentiated phenotype, were not statistically different in carotid arteries containing or deficient in SMC Rac1. Nor did Rac1 deficiency have a statistically significant effect on carotid artery contraction to KCl, a functional readout of the contractile SMC phenotype. Similarly, the abundance of arterial collagen-I, -III, or -V, the integrity of arterial elastin, or arterial responses to pressure, including the axial and circumferential stretchstrain relationships that are assessments of arterial stiffness, were unaffected by deletion of SMC Rac1. Overall, these data argue that the inhibitory effect of Rac1 deletion on in vivo SMC proliferation reflects a primary effect of Rac1 signaling to the cell cycle and is independent of changes in SMC differentiation state or arterial stiffness.