Neutrophils are short-lived, abundant peripheral blood leukocytes that provide a first line of defense against bacterial and fungal infections while also being a key part of the inflammatory response. Chemokines induce neutrophil recruitment to inflammatory sites, where neutrophils perform several diverse functions that are aimed at fighting infections. Neutrophil effector functions are tightly regulated processes that are governed by an array of intracellular signaling pathways and initiated by receptor-ligand binding events. Dysregulated neutrophil activation can result in excessive inflammation and host damage, as is evident in several autoimmune diseases. Rho family small GTPases and agonist-activated phosphoinositide 3-kinases (PI3Ks) represent 2 classes of key regulators of the highly specialized neutrophil. Here we review cross-talk between these important signaling intermediates in the context of neutrophil functions. We include PI3K-dependent activation of Rho family small GTPases and of their guanine nucleotide exchange factors and GTPase activating proteins, as well as Rho GTPase-dependent regulation of PI3K.