Anaplasma phagocytophilum, a member of the family Anaplasmataceae, is the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis. The life cycle of A. phagocytophilum is biphasic, transitioning between the noninfectious reticulate cell (RC) and infectious dense-cored (DC) forms. We analyzed the bacterium's DC surface proteome by selective biotinylation of surface proteins, NeutrAvidin affinity purification, and mass spectrometry. Transcriptional profiling of selected outer membrane protein candidates over the course of infection revealed that aph_0248 (designated asp14 [14-kDa A. phagocytophilum surface protein]) expression was upregulated the most during A. phagocytophilum cellular invasion. asp14 transcription was induced during transmission feeding of A. phagocytophilum-infected ticks on mice and was upregulated when the bacterium engaged its receptor, P-selectin glycoprotein ligand 1. Asp14 localized to the A. phagocytophilum surface and was expressed during in vivo infection. Treating DC organisms with Asp14 antiserum or preincubating mammalian host cells with glutathione S-transferase (GST)-Asp14 significantly inhibited infection of host cells. Moreover, preincubating host cells with GST-tagged forms of both Asp14 and outer membrane protein A, another A. phagocytophilum invasin, pronouncedly reduced infection relative to treatment with either protein alone. The Asp14 domain that is sufficient for cellular adherence and invasion lies within the C-terminal 12 to 24 amino acids and is conserved among other Anaplasma and Ehrlichia species. These results identify Asp14 as an A. phagocytophilum surface protein that is critical for infection, delineate its invasion domain, and demonstrate the potential of targeting Asp14 in concert with OmpA for protecting against infection by A. phagocytophilum and other Anaplasmataceae pathogens.