Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB) in watermelon, a disease that poses a serious threat to watermelon production. Because of the lack of resistant cultivars against BFB, virulence factors or mechanisms need to be elucidated to control the disease. Glycerol-3-phosphate dehydrogenase is the enzyme involved in glycerol production from glucose during glycolysis. In this study, we report the functions of a putative glycerol-3-phosphate dehydrogenase in Ac (GlpdAc) using comparative proteomic analysis and phenotypic observation. A glpdAc knockout mutant, AcΔglpdAc(EV), lost virulence against watermelon in two pathogenicity tests. The putative 3D structure and amino acid sequence of GlpdAc showed high similarity with glycerol-3-phosphate dehydrogenases from other bacteria. Comparative proteomic analysis revealed that many proteins related to various metabolic pathways, including carbohydrate metabolism, were affected by GlpdAc. Although AcΔglpdAc(EV) could not use glu- cose as a sole carbon source, it showed growth in the presence of glycerol, indicating that GlpdAc is involved in glycolysis. AcΔglpdAc(EV) also displayed higher cell- to-cell aggregation than the wild-type bacteria, and tol- erance to osmotic stress and ciprofloxacin was reduced and enhanced in the mutant, respectively. These results indicate that GlpdAc is involved in glycerol metabolism and other mechanisms, including virulence, demon- strating that the protein has pleiotropic effects. Our study expands the understanding of the functions of proteins associated with virulence in Ac.