Synthetic lethality reveals that ROCK1 and PLK1 cooperate in cell migration and invasion. The small Rho-like G-protein, RhoA, is a key regulator of the actin cytoskeleton in cell migration, and is controlled by the RhoA-specific guanine exchange factor, GEF-H1. Here we report that small molecule inhibitors of GEF-H1 synergize with PLK1 inhibitors, such as BI2536, in killing cancer cells that are refractory to BI2536 treatment alone, thus uncovering a synthetic lethal interaction between these proteins in human breast cancer cell lines. The synthetic lethal interaction with PLK1 was associated with defects in actin cytoskeletal rearrangement and abrogation of RhoA activation and migration, implicating GEF-H1/RhoA in PLK1-mediated effects on cell migration and invasion. These data support the use of synthetic lethal approaches to identify pathways that are critical for human cancer cell proliferation, and are necessary for PLK1-dependent regulation of cell migration.