In this paper, MoS2 with S vacancies and Cu loading (Cu/VS-MoS2) was synthesized and used for low-temperature water gas shift reactions (WGSR); the intrinsic reasons and reaction mechanisms for the high catalytic activity of Cu/VS-MoS2 were studied through DFT calculation. The material characterization fully confirms the successful construction of Cu loading and S vacancies on spherical cluster structures of Cu/VS-MoS2. The experimental results show that Cu/VS-MoS2 exhibits the highest CO conversion, up to 40.41%, which is 3.9 times, 1.8 times, and 1.5 times higher than MoS2, VS-MoS2, and Cu/MoS2, respectively. DFT calculations indicate that Cu/VS-MoS2 has high WGSR activity due to the superior electron transfer ability of the dual active sites, and the S vacancy is used to activate CO to generate CO2, while the Cu site activates H2O to produce hydrogen. The synergistic effect of S vacancies and Cu sites, while satisfying hydrogen production and carbon dioxide enrichment, greatly enhances the activity of MoS2. In addition, Cu/VS-MoS2 can more effectively break down the transfer barriers between surface electrons and CO, H2O molecules, allowing electrons to continuously transfer to CO and H2O using S vacancies and Cu atoms as mediators, reducing the energy barrier of the rate-determining step in association mechanism.