Methylphosphonate (MPn), a typical organophosphonate characterized by a C-P bond, profoundly influences phosphorus cycling and methane (CH4) production mechanisms in aquatic ecosystems through its biosynthesis and degradation processes. However, there is limited research on the dynamics of MPn in water bodies and the MPn accumulation capacity of algae. In this study, liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (LC-MS/MS) was employed to quantify MPn in 21 water samples and 15 algal species. Combined with field monitoring, algal laboratory cultivation, and raw water incubation experiments (including MPn/Pi addition, BES treatment, algal filtration, and dark treatment), the relationship between MPn dynamics and CH4 generation was investigated. The results revealed that MPn was detected in 52.4% (11/21) of water samples (1.50±0.24~6.99±0.59 μg/L), and 93.3% (14/15) of algal strains accumulated intracellular MPn (1.87±0.57~22.24±5.81 μg/L). Notably, Microcystis sp. FACHB-3602 exhibited dynamic MPn accumulation during 7 days cultivation (peak value: 8.63±0.85 μg/L), indicating that algae are a significant biological source of MPn in aquatic ecosystems. In both water samples and algae, the contribution of MPn-P to dissolved organic phosphorus (DOP) (0.70%~37.85%、0.21%~0.90%) was significantly higher than that of MPn-C to dissolved organic carbon (DOC) (0.00%~0.05%、0.00%~0.01%), highlighting the dominant role of MPn in phosphorus cycling from an ecological stoichiometric perspective. Raw water incubation experiments demonstrated that MPn addition increased CH4 production by 157.43% compared to the control, while simultaneous addition of inorganic phosphorus (Pi) suppressed CH4 generation. Algal filtration reduced CH4 production by 23.96%, whereas dark treatment promoted CH4 accumulation. These findings suggest that algal-bacterial interactions regulate MPn turnover and aerobic CH4 production, modulated by inorganic phosphorus availability. This study provides critical theoretical insights for further exploration of MPn’s role in aquatic phosphorus cycling and aerobic CH4 production mechanisms.