Submerged aquatic vegetation (SAV) has been successfully used in wetland-based water treatment systems due to its capacity for rapid nutrient uptake, water-column oxygenation, and dampening of sediment resuspension. For Everglades restoration, large engineered wetlands known as Stormwater Treatment Areas (STAs) rely heavily on SAV-dominated treatment cells, since they have been highly efficient at reducing phosphorus (P) in surface waters to mesotrophic levels (12–25 µg/L). However, Everglades STAs have strict water quality effluent limits (∼13 µg/L), and many SAV cells are unable to regularly achieve such levels. In shallow aquatic systems, SAV can be a potential source of internal P loading from sediment to the water column via “nutrient pumping” through root uptake and leaf turnover. This mechanism may be an important obstacle to achieving lower wetland outflow P concentrations, but little is known of the interactions between substrate and SAV nutrition under these low-nutrient conditions.Soil core microcosms were incubated with or without macrophytes to estimate internal P loading potential in an SAV wetland and test the hypothesis that wetland substrate manipulation in the downstream (outflow) region might have reduced such loads and enabled more reliable delivery of low-P discharges. During six sequential two-week batch incubations, water-column P concentrations above muck soil overlain by accrued marl (typical of substrates in existing SAV-dominated STAs) indicated significantly higher internal P loading and higher macrophyte biomass growth than above either a bare limerock (LR) substrate (representative of a 40-ha pilot substrate manipulation study, where the muck soil was removed to expose the underlying limestone bedrock, i.e., “limerock”) or LR with an accrued marl layer (representing post-startup conditions in the pilot, after muck removal and subsequent sediment accumulation under treatment wetland conditions).The results suggest that by removing muck soils to expose a LR substrate in the pilot wetland, SAV biomass and tissue P were diminished, thus reducing the potential for internal loading from SAV, which supports our hypothesis. For each of the three substrate types, internal P loading was higher in the presence of a vascular rooted plant, Potamogeton illinoensis, than the non-vascular macro-alga, Chara sp., (both taxa are prevalent in full-scale STAs), while internal loading was not different between Chara and the non-vegetated control. This indicates the role of P mining by rooted SAV. Overall, this study furthers our understanding of the wetland P cycle at near-oligotrophic levels, and is relevant to STA optimization for Everglades restoration.