ADP-ribosylation factor 6 (ARF6) is a widely expressed GTPase that influences both membrane traffic and actin cytoskeleton function. Its role in dendritic cells (DC) has not previously been investigated. We analysed the effect of retroviral expression of ARF6 GDP/GTP binding and other functional mutants in primary murine DC. Maturation in response to lipopolysaccharide (LPS) proceeded normally in DC expressing ARF6 mutants and production of inflammatory cytokines was similarly unaffected. Although LPS-stimulated macropinocytosis was suppressed by expression of the GTP-binding Q67L ARF6 mutant we detected no overall activation of ARF6 by LPS. The ability of immature DC to migrate towards CCL3 and to a lesser extent, of mature DC to migrate towards CCL19, was compromised by expression of either the Q67L or the GDP-binding T44N mutant. Examination of the actin cytoskeleton in these cells revealed that both mutants strongly inhibited the formation of F-actin-rich podosomes, providing a possible explanation for the effects of ARF6 mutants on DC migration. Thus, these studies identify responses in DC that require normal ARF6 function, though not necessarily further ARF6 activation. They reveal for the first time a role for ARF6 in podosome formation and demonstrate functional effects of the T44N ARF6 mutant.