Seasonal thermal stratification is a common environmental phenomenon in reservoir water bodies and an important research topic for deep water reservoirs. A thermally stratified reservoir is an ideal system for investigating the interaction between factors affecting the environment and microorganism communities under spatial–temporal variation. However, few studies have focused on the effects of water stratification and mixing periods in microbial communities and nitrogen conversion. Therefore, using metagenomic sequencing and quantitative polymerase chain reaction, we investigated the microbial diversity and function in the Xin'anjiang Reservoir during water stratification and mixing period. Principal component analysis indicated that the bacterial communities were mostly affected by water stratification, mainly at 10-m depths, whereas the archaea community was influenced by the entire water profile. The predominant archaeal phyla changed from Candidatus_Woesearchaeota and Euryarchaeota to Thaumarchaeota with the increasing water depth during water stratification. The stratified variations of the dominant archaeal phyla strongly reflected the seasonal changes and vertical gradient in water temperature, dissolved oxygen, and pH. Linear regression analysis indicated that variations in microbial composition altered the overall functional attributes of the communities, especially in the archaeal community. Regarding nitrogen metabolism across the whole reservoir, the number of nitrification and denitrification genes dropped in the same direction as the river flow in spring and summer, while the opposite trend was observed in autumn and winter. The functional genes of the water profiles in the transitional and lacustrine zones varied considerably except in winter. Additionally, the copies of nitrification-related genes were more during the stratification stage than during the mixing stage, whereas copies of denitrification-related genes showed the opposite trend. Overall, this study provides unique insights regarding the effects of seasonal stratification on the distribution and function of microorganisms in large-deep reservoirs, which should be considered when establishing guidelines for reservoirs management.