The small GTPase RhoA regulates the actin cytoskeleton to affect multiple cellular processes including endocytosis, migration and adhesion. RhoA activity is tightly regulated through several mechanisms including GDP/GTP cycling, phosphorylation, glycosylation and prenylation. Previous reports have also reported that cleavage of the carboxy-terminus inactivates RhoA. Here, we describe a novel mechanism of RhoA proteolysis that generates a stable amino-terminal RhoA fragment (RhoA-NTF). RhoA-NTF is detectable in healthy cells and tissues and is upregulated following cell stress. Overexpression of either RhoA-NTF or the carboxy-terminal RhoA cleavage fragment (RhoA-CTF) induces the formation of disorganized actin stress fibres. RhoA-CTF also promotes the formation of disorganized actin stress fibres and nuclear actin rods. Both fragments disrupt the organization of actin stress fibres formed by endogenous RhoA. Together, our findings describe a novel RhoA regulatory mechanism.