Room-temperature sodium-sulfur (RT Na-S) batteries with high energy density and low cost are considered promising next-generation electrochemical energy storage systems. However, their practical feasibility is seriously impeded by the shuttle effect of sodium polysulfide (NaPSs) resulting from the sluggish reaction kinetics. Introducing a suitable catalyst to accelerate conversion of NaPSs is the most used strategy to inhibit the shuttle effect. Traditional catalytic approaches often want to avoid the irreversible phase transition of the catalyst at a deep discharge. On the contrary, here, we leverage the intrinsic structural tunability of the MoS2 catalyst in the opposite direction and innovatively propose a voltage modulation strategy for in situ generation of trace Mo single atoms (MoSAC) during the first charge-discharge process, leading to the formation of highly active catalytic phases (MoS2/MoSAC) through the self-reconstruction. Theoretical calculations reveal that the incorporation of MoSAC modulates the electronic structure of the Mo d-band center, which not only effectively promotes the d-p orbital hybridization but also accelerates the catalytic intermediate desorption by the bonding transition, the dynamic single-atom synergistic catalytic mechanism enhances the adsorption response between the metal active site and NaPSs, which significantly improves the sulfur redox reaction (SRR), and the initial capacity of the MoS2/MoSAC/CF@S cell at 0.2 A g-1 is increased by 46.58% compared to that of the MoS2/CF@S cell. The discovery of the MoS2/MoSAC/CF catalyst provides new insights into adjusting the structure and function of transition metal disulfide catalysts at the atomic scale, offering hope for the development of high-specific-energy RT Na-S batteries.