Lactobacillus is the largest genus within the lactic acid bacteria (LAB), with almost 180 species currently identified. Motility has been reported for at least 13 Lactobacillus species, all belonging to the Lactobacillus salivarius clade. Motility in lactobacilli is poorly characterized. It probably confers competitive advantages, such as superior nutrient acquisition and niche colonization, but it could also play an important role in innate immune system activation through flagellin-Toll-like receptor 5 (TLR5) interaction. We now report strong evidence of motility in a species outside the L. salivarius clade, Lactobacillus curvatus (strain NRIC 0822). The motility of L. curvatus NRIC 0822 was revealed by phase-contrast microscopy and soft-agar motility assays. Strain NRIC 0822 was motile at temperatures between 15°C and 37°C, with a range of different carbohydrates, and under varying atmospheric conditions. We sequenced the L. curvatus NRIC 0822 genome, which revealed that the motility genes are organized in a single operon and that the products are very similar (>98.5% amino acid similarity over >11,000 amino acids) to those encoded by the motility operon of Lactobacillus acidipiscis KCTC 13900 (shown for the first time to be motile also). Moreover, the presence of a large number of mobile genetic elements within and flanking the motility operon of L. curvatus suggests recent horizontal transfer between members of two distinct Lactobacillus clades: L. acidipiscis in the L. salivarius clade and L. curvatus in the L. sakei clade. This study provides novel phenotypic, genetic, and phylogenetic insights into flagellum-mediated motility in lactobacilli.M otility in bacterial species is often mediated by a sophisticated molecular structure called the flagellum. This chief organelle of bacterial motility is self-assembled using dozens of different proteins and rotates to propel the cell forward (1). The filament of the bacterial flagellum is composed of one or more flagellin proteins, a microbe-associated molecular pattern (MAMP) which is recognized by the host Toll-like receptor 5 (TLR5) (2) and which, via activation of the nuclear factor kappa B (NF-B) signaling pathway, engages defense responses both systemically and at epithelial surfaces (3). Several flagellate bacterial pathogens (alphaproteobacteria and epsilonproteobacteria) have evolved flagellin proteins with sequence changes that avoid TLR5 recognition while maintaining motility (4). In an ecosystem, flagellum-mediated motility may confer a competitive advantage on motile species over nonmotile species with respect to niche colonization, biofilm formation, and the secretion of virulence proteins by pathogenic bacteria (5).Lactobacillus spp. constitute a very diverse group and the largest genus within the lactic acid bacteria (LAB). Lactobacilli are associated mainly with food production and probiotics (6). Some Lactobacillus species colonize the gastrointestinal, oral, and genital tracts of humans, making them important members of the human microbiota (7)....