Despite arsenic (As) being very toxic with deleterious effects on metabolism, it can be tolerated and accumulated by some plants. General genetic mechanisms responsible for As tolerance in plants, including Salix species, have been described in transcriptomic analysis, but further experimental verification of the significance of particular transcripts is needed. In this study, a Salix atrocinerea clone, able to thrive in an As-contaminated brownfield, was grown hydroponically in controlled conditions under an As concentration similar to the bioavailable fraction of the contaminated area (18 mg kg^-1) for 30 days. At different time points, i.e. short-term and long-term exposure, biometric data, As accumulation, phytochelatin synthesis, non-protein thiol production and expression of target genes related to these processes were studied. Results showed that S. atrocinerea presents a great tolerance to As and accumulates up to 2400 mg As kg^-1 dry weight in roots and 25 mg As kg^-1 dry weight in leaves. Roots reduce As V to As III rapidly, with As III being the predominant form of As accumulated in root tissues, whereas in the leaves it is As V. After 1 d of As exposure, roots and leaves show de novo synthesis and an increase in non-protein thiols as compared to the control. Integrating these data on As accumulation in the plant and its speciation, non-protein thiol production and the kinetic gene expression of related target genes, a fundamental role is highlighted for these processes in As accumulation and tolerance in S. atrocinerea. As such, this study offers new insights in the plant tolerance mechanisms to As, which provides important knowledge for future application of high-biomass willow plants in phytoremediation of As-polluted soils.