Cancer chemotherapeutics with good anti-migration/anti-metastatic activities are significantly underrepresented in the arsenal of anti-cancer agents. One compound with excellent anti-migration properties in mammalian cell culture is dihydromotuporamine C (Motu 3,3) that was isolated from a sea sponge near Motupore Island, New Guinea. Motu 3,3 has been reported to activate RhoA and to influence sphingolipid metabolism in mammalian cells however the mechanism by which Motu 3,3 inhibits migration in cell culture is not currently understood. Using a leg imaginal disc eversion assay, we show that Motu 3,3 acts through Rho1 in a pathway leading to the activation of the myosin non-muscle heavy chain (Zipper) and the formin actin nucleation (dia) pathway. Additionally, Motu 3,3 activity is antagonized by Rac1. Motu 3,3 exhibits toxicity in cell culture so we tested the mechanism of action of chemical derivatives of Motu 3,3 that have reduced cell culture toxicity but increased ability to inhibit migration. Our data show that the derivatives have the same mode of action as Motu 3,3.