Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, which have the ability to differentiate into fat, cartilage, bone, or nerves that can be applied in tissue engineering. On the other hand, the exoenzyme C3 transferase (C3) is a Rho inhibitor. Once in the cytosol, the cell-penetrating moiety is released, thereby allowing C3 transferase to freely diffuse intracellularly and inactivate RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1. In this study, we investigated ASC cytoskeletal changes induced by the addition of C3 employing immunofluorescence staining, changes in alpha-smooth muscle actin (a-SMA) gene expression employing real-time RT-PCR, and the Rho-inhibitory effect employing the pull-down assay. C3 significantly reduced stress fiber disruption and a-SMA expression 24 h after its addition at a concentration of 1 μg/ml, and it also reduced the Rho activity level. While the correlation of the occurrence can be assumed, it requires further examination to verify it. C3 may be an effective inhibitor of intracellular signal transmission in ASC cytoskeletal control involving Rho.