The impoundment of reservoirs changes the river from a riverine heterotrophic system to a lacustrine autotrophic system, which could be attributed to the shift of pelagic microbial food webs in response to the dam-induced disturbances. However, little is known about what is the key factor controlling this variation and how different underlying interactions affect the food web dynamics. This study investigated the effects of flow velocity and nutrient supply on microbial plankton using a microcosm experiment. The results showed that flow velocity decrease was the main factor inducing the detritus-based food web transformed to the autotroph-based food web, with heterotrophic bacteria and protozoan dominated at high velocity, whereas phytoplankton and metazoan were prevalent in the lentic environment. The lentic-acclimated genera, such as Chlorella sp., Mallomonas sp. and Microcystis sp., showed hysteresis after the velocity recovery, suggesting the potential of algae bloom in reservoirs and even downstream of dams. We further conducted a flow-velocity manipulating experiment and constructed a multi-trophic nitrogen cycling model to provide a mechanistic explanation for the microbial food web dynamics and the nitrogen transformation performances. As indicated in model prediction and sensitivity analysis, the abiotic and biotic variations were directly or indirectly controlled by nutrient utilization and predator-prey interactions. Quantification of these bottom-up and top-down forces revealed the buffer role of predators in mitigating the positive effects of nutrient availability on autotrophs at low velocity and on heterotrophs at high velocity, respectively. This study highlights the importance of mastering the whole information of different trophic levels, in order to better capture the complex microbial food web interactions and the consequent biogeochemical processes in river-reservoir systems.