RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate (PtdIns(3)P) to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of PtdIns(3)P in plants are low, we examined whether Phytophthora species may produce PtdIns(3)P to promote infection. We observed that PtdIns(3)P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae during infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed PtdIns(3)P on their plasma membrane and/or secreted PtdIns(3)P. Silencing of the phosphatidylinositol 3-kinases (PI3K) genes, treatment with LY294002, or expression of PtdIns(3)P-binding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized PtdIns(3)P was required for full virulence. Secretion of PtdIns(3)P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resistance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external PtdIns(3)P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avr1b confirm that both the N-terminus and the C-terminus of this effector can bind PtdIns(3)P.