AbstractOsmotic stress frequently caused by drought is the most threatening environmental stress that remarkably reduces crop yield. Oat (Avena sativa L.) is sensitive to water deficiency during growth. Plant metallothioneins (pMTs) show tremendous promise in enhancing general stress tolerance in plants. This study aimed to verify whether pMTs and elements of the antioxidant defence system protect oat against osmotic stress. Coding and genomic regions of A. sativa L. MTs belonging to three different types were cloned. To evaluate the role of MTs in osmotic stress, the expression of genes encoding AsMT1‐3 was checked by qRT‐PCR in the roots and shoots of oat plants growing in a hydroponic culture in the presence of polyethylene glycol (PEG 6000) and mannitol. The expression of AsMT1‐3 changed in response to osmotic stress; however, the changes depended on the type of MT and the treatment. The amount of AsMT3 transcript was about five‐fold and nine‐fold higher in shoots and roots, respectively, in the presence of mannitol. To further analyse the response of oat to osmotic stress, the level of phenolic compounds, soluble sugars, abscisic acid, and the activity of antioxidant enzymes were tested using spectrophotometric and chromatographic methods. During osmotic stress, the content of phenols, soluble sugars, abscisic acid, and the activity of catalase and peroxidase in shoots increased. In roots treated with PEG 6000, the amount of phenolic compounds was higher than that in roots treated with mannitol. The activity of superoxide dismutase was about 5‐fold higher in roots than in shoots. MTs are involved in plant response to osmotic stress. In the future, insight provided in this study will lead to application in agriculture either by using MTs as molecular markers for stress‐resistant crop cultivars or by a generation of genetically modified crops overexpressing MTs.