Nitrate (NO3 -) contamination in groundwater is a worldwide phenomenon and a pervasive environmental problem, particularly when NO3 --enriched groundwater discharges into a nitrogen-limited estuarine environment through submarine groundwater discharge (SGD). SGD is often associated with eutrophication which ultimately alters the coastal ecology of the receiving surface water. Identifying the sources and transformation processes of NO3 - in and within the groundwater discharged to the estuary provides baseline information underpinning effective management of the coastal environments. The aims of this study were to: (1) understand the linkages between aquifers at different depths and in different parts of a catchment in the south east of Australia which underlies a eutrophic estuary (the Werribee River estuary); (2) identify and apportion the NO3 - source(s) to the aquifers; and (3) identify the major transformation processes of NO3 - in the aquifer along the groundwater flowpath. The average d15N?NO3 - values of the deep groundwater (+21 ?) at the SGD hotspot lies between the enriched d15N?NO3 - ( +33 ?) at the western side of the estuary and relatively depleted d15N?NO3 - ( +14 ?) at the eastern side; indicating that the aquifers from both sides of the estuary are connected at the SGD hotspot. The isotopic composition of NO3 -, together with the concentrations of excess nitrogen (N2) gas also revealed that SGD-derived NO3 - originated predominantly from agricultural activity. Denitrification was not the primary NO3 - removal process in the oxic groundwater. Instead, mixing between sewage (69 ) and fertiliser-derived (31 ) NO3 - appeared to be the main control over the observed NO3 - concentrations ( 1,000 ?mol L-1) and the relatively enriched d15N?NO3 - values (+20 to +23 ?) in the deeper sand and gravel aquifer at the groundwater discharge zone. These results suggest that groundwater is a critical source of NO3 - to the receiving surface water and as such should always be included not only in the regional but also the global N budget. The outcome of the study is a key to sustainable management of coastal aquatic ecosystems.