Mutations in faciogenital dysplasia protein (Fgd1) result in the human disease faciogenital dysplasia (FGDY). Fgd1 contains a RhoGEF domain specific for Cdc42. Fgd1 also contains a Src homology (SH3) binding domain (SH3-BD) that binds directly to the SH3 domain of cortactin, which promotes actin assembly by actin-related protein (Arp)2/3 complex. Here, we report the effect of ligation of cortactin's SH3 domain by the Fgd1 SH3-BD on actin polymerization in vitro. Glutathione S-transferase (GST)-fused Fgd1 SH3-BD enhanced the ability of cortactin to stimulate Arp2/3-mediated actin polymerization. However, a synthetic peptide containing only the SH3-BD sequence had no effect. The SH3-BD peptide bound to cortactin and inhibited the effect of GST-Fgd1 SH3-BD, suggesting that GST dimerization was responsible for the stimulating effect of GST-Fgd1 SH3-BD. When GST-Fgd1 SH3-BD was prepared as a heterodimer with a control GST fusion protein (GST-Pac1), no stimulatory effect on actin polymerization was observed. In addition, when cortactin was dimerized via its N-terminus, away from the C-terminal SH3 domain, actin polymerization with Arp2/3 complex increased markedly, compared to free cortactin. Thus, cortactin ligated by Fgd1 is fully active, indicating that the cell can use Fgd1 to target actin assembly. Moreover, if Fgd1 is multimerized, then cortactin's activity should be enhanced. Fgd1 and cortactin may participate as scaffolds and signal transducers in a positive feedback cycle to promote actin assembly at the cell cortex.