<p>Cold stress has adverse effects on the life of plants by inducing oxidative stress. Specialized metabolites have important roles in stress tolerance of plants. This study was conducted with the aim of investigating the role of sesquiterpene lactones in the reaction of plants to low temperature. Two-month-old <i>Artemisia absinthium</i> in vitro propagated plantlets were exposed to 4 and 10&#xa0;°C as cold and 22&#xa0;°C as control temperatures. Here, we report that sesquiterpene lactones, artemisinin and absinthin, enhance the plant’s tolerance to low temperatures. This enhancement is accompanied by up-regulation of sesquiterpene lactones biosynthetic genes, farnesyl pyrophosphate synthase (<i>FPPS</i>) and germacrene A synthase (<i>GAS</i>), at 4℃ where, <i>MYB4</i> transcript level was also increased as a positive regulator of <i>FPPS</i> expression. Besides, according to the observed increase in malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) contents and also, increased activities of evaluated antioxidative enzymes including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) show that low temperatures induce the oxidative stress in <i>Artemisia absinthium</i> cold treated plantlets. The results also show an osmotic stress following the low temperatures leading to glycine betaine production. In overall, it can be suggested that at moderately low temperatures an osmotic and oxidative stress occurs in <i>Artemisia absinthium</i>. The plant has several mechanisms or approaches to cope the osmotic and oxidative imbalances, and can enhance the specialized metabolites, artemisinin and absinthin, by up-regulation of <i>FPPS</i> and <i>GAS</i> genes which, induced by MYB4 production to promote cold tolerance.</p>