Recently, sulfate-reducing granular sludge has been developed for application in sulfate-laden water and wastewater treatment. However, little is known about biomass stratification and its effects on the bioprocesses inside the granular bioreactor. A comprehensive investigation followed by a verification trial was therefore conducted in the present work. The investigation focused on the performance of each sludge layer, the internal hydrodynamics and microbial community structures along the height of the reactor. The reactor substratum (the section below baffle 1) was identified as the main acidification zone based on microbial analysis and reactor performance. Two baffle installations increased mixing intensity but at the same time introduced dead zones. Computational fluid dynamics simulation was employed to visualize the internal hydrodynamics. The 16S rRNA gene of the organisms further revealed that more diverse communities of sulfate-reducing bacteria (SRB) and acidogens were detected in the reactor substratum than in the superstratum (the section above baffle 1). The findings of this study shed light on biomass stratification in an SRB granular bioreactor to aid in the design and optimization of such reactors.