Profilin-1 (Pfn1 - a ubiquitously expressed actin-binding protein) levels are significantly downregulated in various invasive adenocarcinomas including breast cancer. Although Pfn1 has been shown to be required for motility for most normal cells, breast cancer cells and normal human mammary epithelial cells exhibit a hypermotile phenotype upon Pfn1 depletion, and re-expression of Pfn1 in breast cancer cells decreases their migration. The traditionally conceived pro-migratory function of Pfn1 through its relatively well-studied interactions with actin and polyproline ligands does not provide guidance to explain this context-specific effect of Pfn1 on cell migration. The overall goal of this study is to reveal molecular mechanisms underlying the hypermotile phenotype of breast cancer cells as a result of Pfn1 downregulation. We first show that loss of Pfn1 expression increases motility of breast cancer cells by enhancing targeting of Ena(enabled)/VASP (vasodilator stimulated phosphoprotein) family of actin-binding proteins to the leading edge, a feature that is also reproducible in other cells. We further demonstrate that Ena/VASP targeting to the leading edge is mediated through the action of lamellipodin (Lpd - a membrane anchoring protein) and Pfn1 negatively regulates membrane targeting of Lpd. Limiting Lpd expression impairs motility of Pfn1-deficient breast cancer cells, thereby demonstrating loss of Pfn1 augments breast cancer cell motility through enhanced membrane recruitment of VASP/Lpd complex. Subsequent rescue experiments with various ligand-binding deficient mutants of Pfn1, we further demonstrate that Pfn1 inhibits breast cancer cell motility mainly by its phosphoinositide interaction through negative regulation of Lpd/VASP targeting to the leading edge. Membrane targeting of Lpd in Pfn1-deficient breast cancer cells critically depends on the availability of D3-phosphorylated phosphoinositides, and consistent with this observation, we demonstrate that loss of Pfn1 expression significantly increases PI(3,4)P₂ presentation at the leading edge. Collectively, these findings identify a novel inhibitory mechanism of Pfn1 on breast cancer cell motility by regulating membrane availability of PI(3,4)P₂ and docking of Lpd, and this involves Pfn1’s phosphoinositide interaction. This is in contrast to conventionally thought Pfn1’s regulation of cell motility primarily through its interactions with actin and polyproline ligands.