Production of tumor necrosis factor-α (TNFα) by the neutrophil (PMN) is a pivotal event in innate immunity, but the signals regulating TNFα 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, IκBα degradation, nuclear factor-κB (NF-κB) activation, and induction of TNFα and NF-κB-dependent chemokines. Suppression of TNFα induction by RhoA is Rho kinase α (ROCKα) independent, but Cdc42 dependent, because TNFα induction by C3 transferase is attenuated by inhibition of Cdc42, and constitutively active Cdc42 suffices to activate NF-κB and induce TNFα. 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 ROCKα all promote TNFα protein expression. The p65 subunit of NF-κB 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.