Increasing N concentration and the high density of epiphytic algae are both key factors leading to the decline of submerged macrophytes in many eutrophic lakes. In order to investigate the impacts of increased nitrate-N concentration and the growth of epiphytic algae on the decline of submerged vegetation, we conducted a 2 x 4 factorial experiment with the submerged macrophyte Vallisneria natans (Lour.) Hara by measuring the biomass of plants and some physiological indexes in leaves of V. natans under four nitrate-N concentrations in the water column (0.5, 2.5, 5, and 10 mg/L) and two epiphytic groups (epiphytic algae group and no epiphytic algae group). The results suggested that epiphytic algae could impose adverse effects on the biomass accumulation of V. natans, while the increasing nitrate-N concentration (0.5-10 mg/L) could oppositely promote this process and counteract the adverse effect of epiphytic algae. When nitrate-N concentration was 5 mg/L, the total chlorophyll content in leaves of V. natans in the epiphytic algae group was prominently lower compared with the no epiphytic algae group, while MDA, free proline, and anti-oxidant enzyme (SOD, POD, CAT) activities were significantly higher. Overhigh nitrate-N concentration in the water column also directly imposed adverse effects on the physiology of V. natans. When nitrate-N concentration was over 5 mg/L, the total chlorophyll content and free proline decreased in the no epiphytic algae group, while soluble carbohydrates and soluble proteins decreased when nitrate-N was over 2.5 mg/L. Meanwhile, epiphytic algae and nitrate-N content imposed a synergetic effect on the anti-oxidant enzyme activities of V. natans. When nitrate-N concentration was over 5 mg/L, SOD, POD, and CAT activities kept constant or decreased, which indicated that the oxidation resistance of V. natans was inhibited by stress. Our results indicate that epiphytic algae and increasing nitrate-N concentration in the water column could severally or synergistically impose adverse effects on the physiology of submerged macrophytes and are both key factors leading to the decline of submerged macrophytes.