The Neuroepithelial cell transforming gene 1A (Net1A) is a RhoA subfamily GEF that localizes to the nucleus in the absence of stimulation, preventing it from activating RhoA. Once relocalized in the cytosol, Net1A stimulates cell motility and extracellular matrix invasion. In the present work we investigated mechanisms responsible for Net1A cytosolic relocalization. We demonstrate that inhibition of MAPK pathways blocks Net1A relocalization, with cells being most sensitive to JNK pathway inhibition. Moreover, JNK or p38 MAPK activation is sufficient to elicit Net1A cytosolic localization. Net1A relocalization stimulated by EGF or JNK activation requires nuclear export by CRM1. JNK1 phosphorylates Net1A on serine 52, and alanine substitution of this site prevents Net1A relocalization caused by EGF or JNK activation. Glutamic acid substitution of this site is sufficient for Net1A relocalization and results in elevated RhoA signaling to stimulate myosin light chain phosphorylation and F-actin accumulation. Net1A S52E expression stimulates cell motility, enables Matrigel invasion, and promotes invadopodia formation. These data highlight a novel mechanism for controlling Net1A subcellular localization to regulate RhoA activation, cell motility, and invasion.