ROCK1 and ROCK2 mediate important processes such as cell migration, invasion and metastasis, making them good targets for the development of antitumor agents. Recently, using a fragment-based approach and X-ray crystallography, we reported on the design and synthesis of novel Rho-kinase inhibitors (RKIs). Here, we selected a pair of RKIs, the closely related structural analogs RKI-18 (potent; IC50 values of 397 nM (ROCK1) and 349 nM (ROCK2)) and RKI-11 (weak/inactive; IC50 values of 38 μM (ROCK1) and 45 μM (ROCK2)), as chemical probes and determined their effects on cytoskeleton organization, signaling, apoptosis, anchorage-dependent and independent growth, migration and invasion. RKI-18 but not RKI-11 suppresses potently the phosphorylation of the ROCK substrate myosin light chain 2 (MLC2) in intact human breast, lung, colon and prostate cancer cells. Furthermore, RKI-18 is highly selective at decreasing the levels of P-MLC2 over those of P-Akt, P-S6 and P-Erk ½. RKI-18 suppresses ROCK-mediated actin fiber formation, following stimulation with LPA as well as p21-activated kinase (PAK)-mediated lamellipodia and filopodia formation following bradykinin or PDGF stimulation. Furthermore, RKI-18 but not RKI-11 inhibits migration, invasion and anchorage-independent growth of human breast cancer cells. The fact that the active ROCK inhibitor RKI-18, but not the inactive closely related structural analog RKI-11 is effective at suppressing malignant transformation suggests that inhibition of ROCK with RKI-18 results in preventing migration, invasion and anchorage-independent growth. The potential of this class of RKIs as anti-tumor agents warrants further advanced preclinical studies.