Rivers make vital contributions to the transport of water, sediment and nutrients from terrestrial to marine ecosystems. However, many large rivers worldwide are suffering from dam regulation. Increasing attention has been paid to bacterioplankton communities since they are highly responsive to river alterations and may influence biogeochemical processes. Here, a comprehensive study was conducted in the highly regulated Lancang-Mekong River Basin to address the question of how bacterioplankton communities respond to cascade damming. The results showed that dam constructions increased nutrient concentrations and threatened water quality in cascade reservoirs. Bacterioplankton cell abundance was reduced by damming, and α-diversity was inhibited in cascade reservoirs. Fortunately, however, river ecosystems were resilient after the remarkable disturbance caused by damming. Moreover, bacterioplankton community composition was significantly altered by cascade dams, including a shift in the dominant phylum from r-strategists to k-strategists. Meanwhile, according to GeoChip analysis, the functional composition of bacterioplankton was less affected than taxonomic composition. In addition, geographic and environmental features both followed a distance-decay relationship with community and functional composition, but the local environment condition was the dominant driver in the Lancang River. Therefore, the impoundments of cascade dams had significant impacts on bacterioplankton communities and more attention should be paid to the potential ecological consequences of river regulation.