Neutrophils contain granules loaded with antimicrobial proteins and are regarded as impermeable organelles that deliver cargo via membrane fusion. However, during the formation of neutrophil extracellular traps (NETs), neutrophil elastase (NE) translocates from the granules to the nucleus via an unknown mechanism that does not involve membrane fusion and requires reactive oxygen species (ROS). Here, we show that the ROS triggers the dissociation of NE from a membrane-associated complex into the cytosol and activates its proteolytic activity in a myeloperoxidase (MPO)-dependent manner. In the cytosol, NE first binds and degrades F-actin to arrest actin dynamics and subsequently translocates to the nucleus. The complex is an example of an oxidative signaling scaffold that enables ROS and antimicrobial proteins to regulate neutrophil responses. Furthermore, granules contain protein machinery that transports and delivers cargo across membranes independently of membrane fusion.