Investigations of microplastics have increased exponentially over the past decade, yet no information is currently available on the status of microplastics in strictly regulated, artificial bodies of water. The Middle Route of the South-to-North Water Diversion Project (SNWDP) in China, a highly regulated canal, supplies water to 19 cities and more than 53.10 million residents since 2014, as part of the world's largest inter-basin drinking water diversion project. In this study, the spatiotemporal distribution, characteristics, and polymer types of microplastics were surveyed for the first time in the Middle Route of the SNWDP. On average, microplastics were distributed at abundances of 516 items m(-3) in water, 20 items kg(-1) in biofilms (wet weight), and 24 items kg(-1) in sediments (wet weight), lower than that in other typical inland waters. Both sites and seasons significantly affected microplastic abundance in water, biofilms, and sediments; microplastic abundance in water was also significantly positively correlated with distance to the headwork. The main microplastics in the canal were small (0.05-1 mm) polyethylene tereptahalate (PET) fibers. Interestingly, microplastics were concentrated in biofilms, indicating that biofilms could serve as a sink for microplastics in the canal. Vehicular harvester was used to demonstrate the practicality of biofilm harvest to mitigate contamination with microplastics. Our results showed that microplastics are consistently transported long distances through the canal, biofilms play an important role in the fate of microplastics in the canal, and that biofilm harvest could be potentially used to mitigate microplastic pollution. (C) 2021 Elsevier B.V. All rights reserved.