Sporolactobacillus inulinus has attracted scientific and commercial interest due to its high efficiency in D-lactic acid production. Pyruvate kinase (PYK) is one of the key regulatory points in glycolysis, and well-activated PYK can improve D-lactic acid production. A novel Mn2+/Mg2+-dependent PYK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. Kinetic characterization demonstrated that the S. inulinus PYK had drastically higher activity and affinity toward substrates in the presence of Mn2+ compared to those of the common PYK cofactor Mg2+, and the circular dichroism spectra of the S. inulinus PYK suggested a Mn2+-mediated allosteric activation. The S. inulinus PYK was also allosterically regulated by ribose-5-phosphate or AMP activation and inorganic phosphate or ATP inhibition. The inhibition could be marked reduced or fully eliminated in the presence of activators. The result of fermentations by S. inulinus Y2-8 showed that the extracellular-added MnSO4 and KH2PO4 significantly affected glycolysis flux and D-lactic acid production, which is consistent with the allosteric regulation of Mn2+ and inorganic phosphate on PYK. The sophisticated regulatory role of PYK would establish the foundation of substantial disturbance or restructuring of cellular metabolism for improving the S. inulinus D-lactic acid production.