Increasing evidence indicates that changes in intracellular pH (pHi) regulate cell morphology and cytoskeleton organization. However, our understanding of how pHi affects protein conformations and macromolecular assemblies to regulate cytoskeleton dynamics and other cell behaviors is limited. We are determining design principles, structural features, and functions of pH sensors or proteins that have activities or ligand-binding affinities that are sensitive to small, physiologically relevant changes in pH. Protonation is the simplest possible electrostatic switch for modulating protein function. And in contrast to other posttranslational modifications or cofactors, protonation can confer extremely rapid temporal responses and the modulation of multiple proteins in unison to control a cell behavior. Our current work bridges structural and cellular biology to elucidate at the molecular level how pHi regulates cytoskeletal proteins conferring cell polarity, actin filament assembly, and cell-substrate adhesion remodeling. These processes control distinct stages in directed cell migration, which is dependent on increased pHi in response to migratory cues. Using H+ efflux by the ubiquitously expressed Na-H exchanger NHE1 as a tool to understand pH-dependent cell morphology and cytoskeleton dynamics, we identified the structural basis and functional significance of pH-dependent 1) activity of the GTPase Cdc42 for cell polarity, 2) actin-severing activity by cofilin for de novo actin filament assembly, and 3) talin-actin binding for focal adhesion remodeling. Cdc42 and cofilin activity are regulated by pH-dependent histidine switches for phosphoinositide binding, and talin binding to actin is allosterically regulated by pH. These findings have broad impact on understanding normal epithelial cell biology and pathological processes such as metastatic carcinoma, which is associated with a dysregulated increase in pHi.