The chemotaxis of phosphoinositide kinase-3 (PI3K)-inhibited differentiated HL-60 cells stably expressing CXCR2 was studied in a microfluidic switching gradient device that can generate stable and well-defined forward and reverse gradients. Wortmannin, a widely used PI3K inhibitor, was added during cell preparation and the experiment process. The studies quantify the chemotaxis gradient and the effects of a change in the direction of a CXCL-8 gradient on cell migration. PI3K-inhibited HL-60 cells migrated more efficiently toward the gradient before gradient switching than after, as measured by the effective chemotactic index. The inhibited HL-60 cells also showed that inadequate polarization, slower response time, and reduced cell populations can follow the gradient change. We observed that the role of PI3K in directing cellular response to gradient reversal was important in cell polarization and directional sensing associated with gradient switching.