Significant concentrations of arsenic (As) in mine tailings and surrounding soils suggest that As-contamination is a matter of great concern in the studied mining area. This study aims to evaluate the As contamination, stress responses, tolerance and phytoremediation potential of native flora of As-contaminated tailings in an abandoned mine. The ‘Vale das Gatas’ mine tailings (Northern Portugal) contain high concentrations of As (541–5,770 mg/kg) and heavy metals such as lead, zinc and copper. Average As concentration in colonizing plants, such as Agrostis castellana Boiss. & Reut., range from 13.2 mg/kg in the stems to 30.9 mg/kg in the leaves. Considering the high concentrations of As in the tailings, A. castellana may have developed resistance to these metalloid. In this study, two plant populations of A. castellana were tested in relation to As(V) ion as a possible damaging agent of root membranes in vivo. Stress responses and tolerance to As(V) was tested by a “root growth test” on specimens from the tailings (‘tolerant’), with reference to a population collected outside of the mine influence (‘sensitive’). The tests were performed in hydroponics, for several As(V) concentrations: 10, 20, 40, 80, 160, 320, 640, 1,280 μM. The tolerance index was determined, as the ratio between the average “maximum roots growth” in the presence of As(V) and the average “maximum roots growth” in the absence of As(V). The results allow to verify tolerance indices significantly higher in specimens that grow on the tailings (59.3% maximum), compared with specimens from non-contaminated areas (29.5% maximum). Arsenic concentrations higher than toxic level in A. castellana species, with significant tolerance indices, indicate that internal detoxification tolerance mechanisms might exist. Therefore, this species growing on tailings would be a great advantage in the revegetation of mine areas and can fulfill the objectives of phytostabilization, phytoattenuation, and visual improvement.