The Mekong River in Asia is one of the world's longest rivers. Although it has some of the highest levels of biodiversity and productivity in Asia, the water quality in the basin has recently deteriorated as a consequence of land use changes, dam reservoir construction, population growth, and climate change. For the first time, this study estimates the interannual and monthly variabilities of nutrient fluxes (nitrate - NO3−) and total phosphorus - TP) in the lower Mekong River and Tonle Sap River in Cambodia, and assesses the nutrient linkage between the them. Long-term monitoring data were obtained from Kratie station (in the upper reach of the Mekong River), Chroy Changva station (just upstream of the lower Mekong River–Tonle Sap River confluence), and Prek Kdam station (on the Tonle Sap River ~40 km upstream of the confluence and 70 km downstream of Tonle Sap Lake). From 1995 to 2017, the estimated interannual flux of NO3− was 364 ± 45 kt/y at Kratie and 557 ± 109 kt/y at Chroy Changva. From 2005 to 2017, the estimated interannual flux of TP was 100 ± 16 kt/y at Kratie and 73 ± 19 kt/y at Chroy Changva. Considerable seasonal differences were observed in both fluxes in the Mekong River, with 80–90% of the annual NO3− flux occurring from May to October. The results of the nutrient exchange budget indicated that the NO3− and TP fluxes from the Mekong River into Tonle Sap Lake were ~ 35.8 ± 12.5 kt/y and ~ 8.7 ± 3.3 kt/y, respectively, while these were 34.0 ± 13.8 kt/y and 6.6 ± 1.4 kt/y, respectively, from Tonle Sap Lake into the Mekong system. The results demonstrate that the Mekong River is a vital nutrient source, especially during the flood season, to Tonle Sap Lake and its floodplain.