The study aimed at distinguishing antioxidant responses conditioning acclimation status to metallic stress in a shoot culture of a woody plant Daphne jasminea. Growth, organogenesis, metal accumulation, oxidative stress level and antioxidant responses were compared in the long-term treated shoots (Lt lines) and short-term treated (St lines) with Cd, Ni and Pb in relation to control shoots. Growth rate and organogenic potential were higher in the long-term treated Lt Cd and Lt Pb lines than in respective short-term treated St lines. Lt Ni line proliferated less effectively than St Ni line, whereas biomass accretion was similar in both Ni lines. All heavy metal (HM)-exposed shoots suffered from oxidative stress, however, it was more severe in St than in Lt lines. In the long-term treated lines three major factors were associated with the acclimation status: activity of peroxidase (POD) and accumulation of anthocyanins and carotenoids. POD activity was 4-1.5-times higher in Lt lines than in the control one. Anthocyanin content nearly tripled, while carotenoid content doubled in Lt lines. Other elements of antioxidant system, CAT and SOD, glutathione, ascorbic acid and proline, complemented the action of the "major players" in long-term treated Lt lines depending on heavy metal type. Short-term treated lines responded by accumulation of proteins and a decline in catalase activity. The study demonstrated that long-term heavy metal treatment can be considered acclimation strategy, during which substantial modulation of the antioxidant system occurs. Due to orchestrated action of cytosolic POD, vacuolar anthocyanins and membrane-bound carotenoids HM toxicity could be counteracted in various cellular compartments. Key message Acclimation to heavy metal exposure in D. jasminea shoot cultures involves modulation of antioxidant activity and is conditioned by an orchestrated action of cellular peroxidases, vacuolar anthocyanins and membrane-bound carotenoids.