For centuries, many Mediterranean catchments were covered with vineyards in which copper was widely applied to protect grapevines against fungus. In the Mediterranean-type flow regime, brief and intense flood events increase the stream water discharge by up to 10 times and cause soil leaching and storm runoff. Because vineyards are primarily cultivated on steep slopes, high Cu fluxes are discharged by surface water runoff into the rivers. The purpose of this work was to investigate the riverine behavior and transport of anthropogenic Cu by coupling a sequential chemical extraction (SCE) procedure, used to determine Cu partitioning between residual and non-residual fractions, with δ65Cu isotopic measurements in each fraction. In the Baillaury catchment, France, we sampled soils (cultivated and abandoned), river bed sediments (BS), suspended particulate matter (SPM), and river water during the flash flood event of February 2009. Copper partitioning using SCE show that most of Cu in abandoned vineyard soil was in the residual phase (>60%) whereas in cultivated soil, BS and SPM, Cu was mostly (>25%) in non-residual fractions, mainly adsorbed onto iron oxide fractions. A small fraction of Cu was associated with organic matter (5 to 10%). Calculated enrichment factors (EF) are higher than 2 and the anthropogenic contribution was estimated between 50 to 85%. Values for δ65Cu in bulk samples were similar to bedrock therefore; δ65Cu on SCE fractions of superficial soils and SPM allowed for discrimination between Cu origin and distribution. Copper in residual fractions was of natural mineral origin (δ65Cu close to local bedrock, +0.07‰). Copper in water soluble fraction of SPM (δ65Cu = +0.26‰) was similar to dissolved river Cu (δ65Cu = +0.31‰). Copper from fungicide treatment (δ65Cu = −0.35‰) was bound to organic matter (δ65Cu = −0.20‰) without or with slight isotopic fractioning. A preferential adsorption of 65Cu onto iron oxides (δ65Cu = +0.5‰) is shown.