A new technique for the determination of dissolved copper isotope composition (δ65Cu) of seawater was applied to examine copper sources and internal cycling in the Mediterranean Sea. A succession of chelating resin with nitrilotriacetic acid functional groups and strong base anion exchange resin, together with optimization of the multi-collector inductively coupled plasma mass spectrometry set-up allowed to isolate copper from seawater matrix and to measure the 65Cu/63Cu ratios in seawater with an external precision of 0.06‰ (2 s.d.). This method was first applied for inter-comparison measurements to surface and deep waters sampled at station BATS in the North Atlantic Ocean. Disparities in δ65Cu reported here and in the literature over these samples showed a need to investigate whether a new inter-comparison sample should be decided upon, or whether the use of UV-oxidation can also affect the measurement of δ65Cu. We also measured total dissolved Cu concentration ([dCu]T) and δ65Cu for 12 stations in the Mediterranean Sea. The δ65Cu distribution showed significant variations in the euphotic zone, at the Chl a maximum and at bottom depths. Copper isotope ratios ranged from +0.21‰ to +0.76‰ (±0.06‰, 2 s.d.), yielding an average of +0.51‰ (±0.20‰, 2 s.d.; n = 96) for the Mediterranean Sea. A strong zonation between natural aerosol deposition to the South and anthropogenic aerosol deposition to the North was seen in the sample set (Dulaquais et al. 2017; Gerringa et al. 2017; Rolison et al. 2015). Natural dust deposits seemed to draw δ65Cu toward lower values and deeper in the euphotic zone whereas the impact of anthropogenic aerosols seemed restricted to an increase in [dCu]T. At the Chl a maximum, δ65Cu showed significant increase which we attributed to scavenging on and/or uptake by phytoplankton. The isotope signature of Cu sources to the Mediterranean Sea were also investigated and we inferred a significant source of isotopically heavy Cu in the Gibraltar area, potentially originating from a release of Cu sulfide leached in the mining area of Southern Spain (i.e. Iberian Pyrite Belt) in rivers and transported to the Mediterranean Sea in surface by seasonal water mass circulation. Conversely, marine sediments appear to be a source of isotopically-light Cu to deep waters. This study provides new hints on the use of copper isotopes to study sources and sinks of Cu in seawater. This article is part of a special issue entitled: Conway GEOTRACES - edited by Tim M. Conway, Tristan Horner, Yves Plancherel, and Aridane G. González.