Production of tumor necrosis factor-alpha (TNFalpha) by the neutrophil (PMN) is a pivotal event in innate immunity, but the signals regulating TNFalpha induction in this primary cell are poorly understood. Herein, we use protein transduction to identify novel, opposing anti- and pro-cytokine-inducing roles for RhoA in the resting and lipopolysaccharide (LPS)-stimulated human PMN, respectively. In the resting cell, RhoA suppresses Cdc42 activation, IkappaBalpha degradation, nuclear factor-kappaB (NF-kappaB) activation, and induction of TNFalpha and NF-kappaB-dependent chemokines. Suppression of TNFalpha induction by RhoA is Rho kinase alpha (ROCKalpha) independent, but Cdc42 dependent, because TNFalpha induction by C3 transferase is attenuated by inhibition of Cdc42, and constitutively active Cdc42 suffices to activate NF-kappaB and induce TNFalpha. By contrast, we also place RhoA downstream of p38 mitogen-activated protein kinase and Cdc42 in a novel LPS-activated pathway in which p38, Cdc42, and ROCKalpha all promote TNFalpha protein expression. The p65 subunit of NF-kappaB coprecipitates with RhoA in a manner sensitive to the RhoA activation state. Our findings suggest a new, 2-faced role for RhoA as a checkpoint in innate immunity.