Abstract Dendritic cell (DC) migration is essential for efficient host defense against pathogens and cancer, as well as for the efficacy of DC-based immunotherapies. However, the molecules that induce the migratory phenotype of DCs are poorly defined. Based on a large-scale proteome analysis of maturing DCs, we identified the GPI-anchored protein semaphorin 7A (Sema7A) as being highly expressed on activated primary myeloid and plasmacytoid DCs in human and mouse. We demonstrate that Sema7A deficiency results in impaired chemokine CCL21-driven DC migration in vivo. Impaired formation of actin-based protrusions, resulting in slower three-dimensional migration, was identified as the mechanism underlying the DC migration defect. Furthermore, we show, by atomic force microscopy, that Sema7A decreases adhesion strength to extracellular matrix while increasing the connectivity of adhesion receptors to the actin cytoskeleton. This study demonstrates that Sema7A controls the assembly of actin-based protrusions that drive DC migration in response to CCL21.