Actin polymerization in the cytosol and at the plasma membrane is locally regulated by actin nucleators. Several microbial pathogens exploit cellular actin polymerization to spread through tissue. The movement of the enteric pathogen Shigella flexneri, both within the cell body and from cell to cell, depends on actin polymerization. During intercellular spread, actin polymerization at the bacterial surface generates protrusions of the plasma membrane, which are engulfed by adjacent cells. In the cell body, polymerization of actin by Shigella spp. is dependent on N-WASP activation of the Arp2/Arp3 complex. Here we demonstrate that, in contrast, efficient protrusion formation and intercellular spread depend on actin polymerization that involves activation of the Diaphanous formin Dia. While the Shigella virulence protein IpgB2 can bind and activate Dia1 (N. M. Alto et al., Cell 124:133-145, 2006), its absence does not result in a detectable defect in Dia-dependent protrusion formation or spread. The dependence on the activation of Dia during S. flexneri infection contrasts with the inhibition of this pathway observed during vaccinia virus infection.