AbstractFilamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogenPhytophthora infestansenter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing ofNbCHC, encoding clathrin heavy chain, or the endosome marker geneNbAra6encoding a Rab GTPase in the model hostNicotiana benthamiana, attenuatedP. infestansinfection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencingPP1cisoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that the uptake ofP. infestansRXLR effectors into plant cells occurs via CME.