Costal eutrophication leads to increased sulfide levels in sediments, which has been identified as a major cause of the global decline in seagrass beds. The seagrass Thalassia hemprichii, a dominant tropical species in the Indo-Pacific, is facing a potential threat from sulfide, which can be easily reduced from sulfate in porewater under the influence of global climate change and eutrophication. However, its metabolic response and tolerance mechanisms to high sulfide remain unclear. Thus, the current study investigated the physiological responses and programmed metabolic networks of T. hemprichii through a three-week mesocosm experiment, integrating physiology, stable isotope, widely targeted metabolomics, transcriptomics, and microbial diversity assessments. High sulfide reduced the sediment microbial diversity, while increased sediment sulfate reduced bacterial abundance and δ34S. The exposure to sulfide enhanced root δ34S while decreased leaf δ34S in T. hemprichii. High sulfide was shown to inhibit photosynthesis via damaging PSII, which further reduced ATP production. In response, abundant up-regulated differentially expressed genes in energy metabolism, especially in oxidative phosphorylation, were activated to compensate high energy requirement. High sulfide also promoted autophagy by overexpressing the genes related to phagocytosis and phagolysosome. Meanwhile, metabolomic profiling revealed that the contents of many primary metabolites, such as carbohydrates and amino acids, were reduced in both leaves and roots, likely to provide more energy and synthesize stress-responsive secondary metabolites. Genes related to nitrate reduction and transportation were up-regulated to promote N uptake for sulfide detoxification. High sulfide levels specifically enhanced thiamine in roots, while increased jasmonic acid and flavonoid levels in leaves. The distinct differences in metabolism between roots and leaves might be related to sulfide levels and the growth-defense trade-off. Collectively, our work highlights the specific mechanisms underlying the response and tolerance of T. hemprichii to high sulfide, providing new insights into seagrass strategies for resisting sulfide.