The activity of allosterically regulated enzyme is modulated through structural changes induced by effectors. L-lactate dehydrogenase from Geobacillus stearothermophilus (GsLDH) catalyzes the reversible conversion between pyruvate and lactate using NAD(H), and its activity is known to be activated by fructose 1,6-bisphosphate (FBP). However, the molecular basis of this regulation has not been explored using molecular dynamics (MD) simulations. In this study, we integrated MD simulations with biochemical assays to investigate the impact of FBP on GsLDH structure and function. MD revealed that FBP stabilizes the tetrameric form, reduces residue flexibility, and enhances pyruvate interactions with active site residues, despite a 23 Å distance between binding sites. Using MDavocado, we identified three P-axis-related dimer interface regions critical for stability and structural integrity. Microcalorimetry titration revealed that NADH binding (Kd = 1.2 ± 0.3 μM) occurs only in the presence of FBP, indicating an enthalpy-driven interaction involving a hydrogen-bond network. Single amino acid replacement, Gln189Leu, maintains tetrameric structure without FBP and enhances the substrate inhibition effect. However, this mutant fails to trigger the allosteric transition toward a conformation with higher affinity for the substrate, resulting in a high K0.5 value (2.3 ± 0.2 mM) and a low kcat/K0.5 (32 ± 4 s−1 mM−1), comparable to that of the WT without FBP. These findings suggest that oligomerization alone does not confer allosteric responsiveness, emphasizing the essential role of specific interactions in allosteric regulation. Collectively, our results advance the molecular understanding of FBP as a key allosteric effector to stabilize quaternary structure and improve enzyme activity.