ABSTRACTSpiroplasmabacteria are highly motile bacteria with no cell wall and a helical morphology. This clade includes many vertically transmitted insect endosymbionts, includingSpiroplasma poulsonii, a natural endosymbiont ofDrosophila melanogaster.S. poulsoniibacteria are mainly found in the hemolymph of infected female flies and exhibit efficient vertical transmission from mother to offspring. As is the case for many facultative endosymbionts,S. poulsoniican manipulate the reproduction of its host; in particular,S. poulsoniiinduces male killing inDrosophila melanogaster. Here, we analyze the morphology ofS. poulsoniiobtained from the hemolymph of infectedDrosophila. This endosymbiont was not only found as long helical filaments, as previously described, but was also found in a Y-shaped form. The use of electron microscopy, immunogold staining of the FtsZ protein, and antibiotic treatment unambiguously linked the Y shape ofS. poulsoniito cell division. Observation of the Y shape in anotherSpiroplasma,S. citri, and anecdotic observations from the literature suggest that cell division by longitudinal scission might be prevalent in theSpiroplasmaclade. Our study is the first to report the Y-shape mode of cell division in an endosymbiotic bacterium and addsSpiroplasmato the so far limited group of bacteria known to utilize this cell division mode.IMPORTANCEMost bacteria rely on binary fission, which involves elongation of the bacteria and DNA replication, followed by splitting into two parts. Examples of bacteria with a Y-shape longitudinal scission remain scarce. Here, we report thatSpiroplasma poulsonii, an endosymbiotic bacterium living inside the fruit flyDrosophila melanogaster, divide with the longitudinal mode of cell division. Observations of the Y shape in anotherSpiroplasma,S. citri, suggest that this mode of scission might be prevalent in theSpiroplasmaclade.Spiroplasmabacteria are wall-less bacteria with a distinctive helical shape, and these bacteria are always associated with arthropods, notably insects. Our study raises the hypothesis that this mode of cell division by longitudinal scission could be linked to the symbiotic mode of life of these bacteria.