To understand the longitudinal variation in the structure and functioning of large river ecosystems, six Ecopath models were constructed to exhibit the energy flows of aquatic food webs along the subtropical East River in China. Input parameters were primarily obtained from field data collected in 2012–2016; model outputs were estimated by network analysis. Longitudinally, ecosystem characteristics of the East River had high spatial heterogeneity. The biomass, production, and consumption, of which >75.8% of each was distributed at trophic level (TL) II, were determined by aquatic insects upstream, molluscs midstream, and zooplankton downstream. Carnivorous fish occupied the maximal TL of 2.95–3.50. Due to the different trophic interactions of regional food webs, the keystone groups shifted from odonate larvae in headwaters to piscivorous fish upstream/midstream, to zooplankton downstream, and to phytoplankton near the estuary. Aquatic insects, insectivorous fish, epiphytes, and hydrophytes, all of which had ecotrophic efficiencies >0.99, were critical groups that influenced mass balance through short prey supply. The mean transfer efficiencies (TEs) through TLs IIIV had the lowest values of 1.8–4.1% upstream and increased to the highest levels of 8.0–8.4% midstream before they decreased to 6.4–7.0% downstream. The low TEs along the river were limited mainly by the lack of carnivorous fish upstream and the low predation on mollus and plankton midstream/downstream. A series of theory and information indices showed that the pristine upstream system was mature but underdeveloped in organisation; in contrast, the downstream systems in the urban and industrial reaches were immature and stressed in terms of excessive phytoplankton production and short cycling.