In E. coli and P. ananatis, L-serine biosynthesis is initiated by the action of D-3-phosphoglycerate dehydrogenase (SerA), which converts D-3-phosphoglycerate into 3-phosphohydroxypyruvate. SerA can concomitantly catalyze the production of D-2-hydroxyglutarate (D-2-HGA) from 2-ketoglutarate by oxidizing NADH to NAD+. Several bacterial D-2-hydroxyglutarate dehydrogenases (D2HGDHs) have recently been identified, which convert D-2-HGA back to 2-ketoglutarate. However, knowledge about the enzymes that can metabolize D-2-HGA is lacking in bacteria belonging to the Enterobacteriaceae family. We found that ydiJ encodes novel D2HGDHs in P. ananatis and E. coli, which were assigned as D2HGDHPa and D2HGDHEc, respectively. Inactivation of ydiJ in P. ananatis and E. coli led to the significant accumulation of D-2-HGA. Recombinant D2HGDHEc and D2HGDHPa were purified to homogeneity and characterized. D2HGDHEc and D2HGDHPa are homotetrameric with a subunit molecular mass of 110 kDa. The pH optimum was 7.5 for D2HGDHPa and 8.0 for D2HGDHEc. The Km for D-2-HGA was 208 μM for D2HGDHPa and 83 μM for D2HGDHEc. The enzymes have strict substrate specificity towards D-2-HGA and displayed maximal activity at 45 °C. Their activity was completely inhibited by 0.5 mM Mn2+, Ni2+ or Co2+. The discovery of a novel family of D2HGDHs may provide fundamental information for the metabolic engineering of microbial chassis with desired properties.