Long periods of high temperature or transitory increased temperature, a widespread agricultural problem, may lead to a drastic reduction in economic yield, affecting plant growth and development in many areas of the world. Heat stress causes many anatomical and physiological changes in plants. Its unfavorable effects can be alleviated by thermotolerance induced by exogenous application of plant growth regulators and osmoprotectants or by gradual application of temperature stress. Digitalis trojana Ivanina is an important medicinal plant species well known mainly for its cardenolides. The production of cardenolides via traditional agriculture is commercially inadequate. In this study, elicitation strategies were employed for improving crop thermotolerance and accumulation of cardenolides. For these purposes, the effects of salicylic acid (SA) and/or high temperature treatments in inducing cardenolide accumulation and thermotolerance were tested in callus cultures of D. trojana. Considerable increases in the production of cardenolides (up to 472.28 μg.g−1 dry weight, dw) and induction of thermotolerance capacity were observed when callus cultures were exposed to high temperature for 2 h after pretreating with SA. High temperature treatments (2 h and 4 h) caused a marked reduction in superoxide dismutase (SOD; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6) activities, while SA pretreatment increased their activities. High temperature and/or SA appeared to increase the levels of proline, total phenolic, and flavonoid content. Elevated phenolic accumulation could be associated with increased stress protection. These results indicated that SA treatments induced synthesis of antioxidants and cardenolides, which may play a significant role in resistance to high temperature stress. •SA alleviates inhibitory effects of heat stress.•SA induces thermotolerance and cardenolide accumulation in Digitalis trojana.•SA activated antioxidant responses in D. trojana.