Neorickettsia sennetsu is an obligate intracellular bacterium of monocytes and macrophages and is the etiologic agent of human Sennetsu neorickettsiosis. Neorickettsia proteins expressed in mammalian host cells, including the surface proteins of Neorickettsia spp., have not been defined. In this paper, we isolated surface-exposed proteins from N. sennetsu by biotin surface labeling followed by streptavidin-affinity chromatography. Forty-two of the total of 936 (4.5%) N. sennetsu open reading frames (ORFs) were detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS), including six hypothetical proteins. Among the major proteins identified were the two major β-barrel proteins: the 51-kDa antigen (P51) and Neorickettsia surface protein 3 (Nsp3). Immunofluorescence labeling not only confirmed surface exposure of these proteins but also showed rosary-like circumferential labeling with anti-P51 for the majority of bacteria and polar to diffuse punctate labeling with anti-Nsp3 for a minority of bacteria. We found that the isolated outer membrane of N. sennetsu had porin activity, as measured by a proteoliposome swelling assay. This activity allowed the diffusion of L-glutamine, the monosaccharides arabinose and glucose, and the tetrasaccharide stachyose, which could be inhibited with anti-P51 antibody. We purified native P51 and Nsp3 under nondenaturing conditions. When reconstituted into proteoliposomes, purified P51, but not Nsp3, exhibited prominent porin activity. This the first proteomic study of a Neorickettsia sp. showing new sets of proteins evolved as major surface proteins for Neorickettsia and the first identification of a porin for the genus Neorickettsia.