Gracilariopsis lemaneiformis is a kind of cultivated seaweed with the second highest production in China, which is mainly used to feed abalone and extract agar, but its yield and cultivated period are limited due to the high temperature of the summer in the southern coast. As a signaling molecule, non-reducing disaccharide trehalose plays an important role in enduring high temperature, drought and other adverse stresses. To explore the role and mechanism of trehalose in the stress resistance of G. lemaneiformis, this paper investigated the effects of exogenous trehalose on the growth, stress resistance-related substances, metabolic enzymes, endogenous salicylic acid and related genes under high temperature stress using physiological and biochemical test, HPLC-MS/MS and real-time fluorescent quantitative PCR (qRT-PCR). The results showed that application of 10 mmol/L trehalose promoted the algal growth and proline content of G. lemaneiformis with 0.34-fold (3 d) and 0.30-fold increases(24 h) compared to the control, respectively. Meanwhile, trehalose significantly activated the chlorophyll fluorescence parameters and the activities of superoxide dismutase and trehalose-6-phosphate synthase, but malonaldehyde content and lipoxidase (LOX) activity were reduced. Moreover, trehalose significantly stimulated the activity and gene expression of isochorismate synthase, the key enzyme in salicylic acid biosynthesis, and the accumulation of salicylic acid (2.05-fold). Finally, supplementation with trehalose could maintain the stable increase of the gene expression level of heat shock protein 70, while the lox2 gene expression was downregulated at 12 h. All of these results revealed that trehalose could promote algal growth through scavenging the reactive oxygen species, alleviating the lipid peroxidation of the cell membrane, enhancing the accumulation of osmolytes and phytohormones and so on. This study verifies that exogenous trehalose plays a vital role in resisting high temperature, which will provide a reference for future breeding efforts to improve the stress resistance of G. lemaneiformis.