Despite recent advances in our understanding of biochemical regulation of neutrophil chemotaxis, little is known about how mechanical factors control neutrophils’ persistent polarity and rapid motility. Here, by using a human neutrophil-like cell line and human primary neutrophils, we describe a dynamic spatiotemporal pattern of tractions during chemotaxis. Tractions are located at both the leading and the trailing edge of neutrophils, where they oscillate with a defined periodicity. Interestingly, traction oscillations at the leading and the trailing edge are out of phase with the tractions at the front leading those at the back, suggesting a temporal mechanism that coordinates leading edge and trailing edge activities. The magnitude and periodicity of tractions depend upon the activity of non-muscle myosin IIA. Specifically, traction development at the leading edge requires myosin light chain kinase (MLCK)-mediated myosin II contractility and is necessary for 51- integrin activation and leading edge adhesion. Localized myosin II activation induced by spatially activated small GTPase Rho and its downstream kinase p160-ROCK, as previously reported, leads to contraction of actin-myosin II complexes at the trailing edge, causing it to de-adhere. Our data identify a key biomechanical mechanism for persistent cell polarity and motility.