<p>Carbon/nitrogen (C/N) ratios and carbon isotopic compositions (δ¹³C) are widely used to trace organic matter sources and to reconstruct paleoenvironments. The C/N ratio and δ¹³C proxies of submerged aquatic plants are often considered to have a specific value. However, these indicators may be influenced by catchment land use that affects the water chemistry. Variable C/N ratios and δ¹³C values of end-members may introduce biases that affect the tracing of organic matter sources and paleoenvironmental reconstructions. We conducted controlled mesocosm experiments using five artificial ponds containing cultured charophytes of the same species but grown under different catchment land-use types. Charophyte elemental contents (C, N), C/N ratios, and dual-carbon isotopes (δ¹³C–Δ¹⁴C) were analyzed, alongside with the water chemistry (DIC, NO<Stack> ₃ ⁻ </Stack>). Whole plant C, N contents decreased due to CaCO₃ encrustation, and decarbonated plant revealed consistent organic C ∼44%–45% in different pond systems. Plant C/N ratios differed significantly among systems, ranging from 13.5 to 22.1. Decarbonated plants showed strong negative correlation between C/N and tissue N content, driven by water NO<Stack> ₃ ⁻ </Stack> availability mainly. Lower N concentrations under nutrient-poor conditions increased C/N ratio. δ¹³C values spanned from −15.2‰ to −28.0‰, while Δ¹⁴C ranged from −91‰ to −253‰. These variations strongly correlated with the δ¹³C and Δ¹⁴C of DIC. Catchment land-use was shown to regulate the chemical composition of submerged plants by altering the water chemistry. This study shows that high C/N ratios and depleted δ¹³C values in aquatic organic matter do not necessarily mean that the organic matter originates from terrestrial plants, as it may come from autochthonous aquatic plants. The variable C/N ratios and δ¹³C values in aquatic plants complicate the tracing of organic matter and subsequent paleoenvironmental reconstruction using traditional criteria.</p>