Incorporation of rice straw to soil is a common agricultural practice in rice cultivation. In anaerobic paddy soil, the complete mineralization of organic matter to CH4 and CO2 is accomplished by the sequential reduction of nitrate, ferric iron, sulfate, and methanogenesis. In order to estimate the temporal changes of sulfate-reducing prokaryotes (SRP) as decomposers of organic matters, the effects of rice straw amendment on the dynamics of sulfate reduction and SRP were investigated by combining the monitoring of CH4, sulfate, and organic acids with molecular tools such as soil DNA extraction, real-time PCR, cloning, sequencing, and phylogenetic analysis. The incorporation of rice straw into paddy soil significantly increased concentrations of sulfate, formate, acetate, propionate, and lactate and CH4 production. The rate of sulfate reduction in the straw-amended slurries was significantly higher than that in the unamended slurries. The dsrAB gene copy numbers of SRP in the straw-amended soil slurries ranged from 4.26 × 106 to 1.96 × 108 per gram of dry soil, which were significantly higher than those in the unamended control ranging from 1.99 × 106 to 7.90 × 107 per gram of dry soil. Significant correlations were observed between SRP dsrAB gene copy numbers and the concentrations of sulfate and acetate. Cloning and sequencing analyses showed a clear shift of SRP community structure between treatments and time. In the straw-amended slurries, Clostridia-like SRP significantly increased, while Deltaproterobacteria-like SRP (Sytrophobacter, Desulfobacterium, Desulfovibrio, and Desulfomonile) decreased during the incubation period. Novel uncultured SRP were abundant in the straw-amended slurries and changed during the incubation period.