Rahnella aquatilis HX2 (proteobacteria) shows tolerance to selenium (Se). The minimum inhibitory concentrations of selenomethionine (Se-Met), selenite [Se (IV)], and selenate [Se (VI)] to HX2 are 4.0, 85.0, and 590.0 mM, respectively. HX2 shows the ability to reduce Se (IV) and Se (VI) to elemental Se nanoparticles (SeNPs). The maximum production of SeNPs by HX2 strain is 1.99 and 3.85 mM in Luria-Bertani (LB) broth with 5 mM Se (IV) and 10 mM Se (VI), respectively. The morphology of SeNPs and cells were observed by transmission electron microscope, environmental scanning electron microscope, and selected area electric diffraction detector. Spherical SeNPs with amorphous structure were found in the cytoplasm, membrane, and exterior of cells. Morphological variations of the cell membrane were further confirmed by the release of cellular materials absorbed at 260 nm. Flagella were inhibited and cell sizes were 1.8-, 1.6-, and 1.2-fold increases with the Se-Met, Se (VI), and Se (IV) treatments, respectively. The real-time quantitative PCR analysis indicated that some of the genes controlling Se metabolism or cell morphology, including cysA, cysP, rodA, ZntA, and ada, were significantly upregulated, while grxA, fliO, flgE, and fliC genes were significantly downregulated in those Se treatments. This study provided novel valuable information concerning the cell morphology along with biological synthesis process of SeNPs in R. aquatilis and demonstrated that the strain HX2 could be applied in both biosynthesis of SeNPs and in management of environmental Se pollution.