Objective To determine whether the alteration of cytoskeleton interfere with the mechanical properties of normal rat kidney cells (NRKCs).Method NRKCs were treated with cytochalasin D, colchicine and blebbistatin using micropipette aspiration technique, and the changes in Young’s modulus and viscoelasticity of NRKCs were assessed. Results The Young's modulus of is significantly decreased after treated with all cytoskeleton inhibitors. Compared to the control, the viscoelasticity is significantly decreased in NRKCs treated with cytochalasin D and blebbistatin, but in not in colchicinestreated cells. Conclusions The effect of blebbistatin on NRKCs mechanical properties is independent of cytoskeleton. Inhibition of assembling of actin meshwork or the activity of myosin II ATPase will reduce cell's Young's modulus and viscoelasticity. In addition, microtubule assembling is correlated with Young's modulus rather than viscoelasticity.