A bio-rack system was developed for treating Cu-contaminated freshwaters. Each pilot constructed wetland (CW, 110 dm3) contained 15 perforated vertical pipes filled with a mixture of gravel (diorite; 80%) and perlite (20%) and assembled as a rack. The whole experimental device consisted of 12 CW planted either with Phragmites australis, Phalaris arundinacea or Juncus articulatus, and unplanted as control (in triplicates). All plants were sampled at a Cu-contaminated site. The CWs were filled with a mix of freshwater (30%) from the Jalle d’Eysines River (Bordeaux, France) and tap water (70%). Water was spiked with Cu (2.5 μM, 158.5 μg L−1). Three CW batches were carried out, i.e. in early spring (March, S#1), beginning of the growing season (May, S#2), and peak growing season (June, S#3). The S#3 water was initially acidified to pH 6. For all batches, water was recirculated in the CW during 14 days. Physico-chemical parameters (pH, electrical conductivity, redox potential, BOD5 and Cu2+ concentrations) were measured every three days. Water pH of both S#1 and #2 ranged between 7.8 and 8.5 for all treatments during the experiment. Initial and final total Cu concentrations were analysed for all CWs and batches. Relative Treatment Efficiency Index (RTEI) indicated the plant effect compared to the unplanted CW. Free Cu2+ removal was <10% for all S#1 treatments (RTEI ranged between 0 and −1) whereas it increased to 77% (RTEI = 0.1) in S#2 for P. arundinacea. In acidic conditions (S#3), Cu2+ removal was 99% for all treatments (RTEI = 0). For S#1 and S#2, highest total Cu removal occurred in CW planted with P. arundinacea (respectively 52% and 68%, RTEI = 0.1 and 0.2). For S#3, total Cu removal peaked up to 90% in the unplanted CW. The RTEI values suggested no beneficial effect of macrophytes on Cu removal at short term. Conversely, the CW planted with J. articulatus generally displayed a lower efficiency. The lowest value for total Cu concentration in water after the 14-day period was 13 μg L−1 in S#3 unplanted and planted with P. arundinacea. The role of the biofilm as a key-player of Cu removal in such bio-racks is discussed.