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Horseshoe crabs, the most well-known example of "living fossils", are iconic and ecologically important macroinvertebrates in coastal and estuarine ecosystems. Their blood is a crucial resource for manufacturing Limulus or Tachypleus amebocyte lysate to detect bacterial endotoxins or fungal contamination in drug and medical devices. An enhanced understanding of their ecological roles and trophic interactions in the food webs is fundamental to facilitate resource management for the declining populations in Asia. Foraging information of the Asian species, however, is mainly derived from preliminary, scattered reports from a limited number of study locations. In this study, resource utilization, trophic niche dynamics, and trophic interaction of the juvenile tri-spine horseshoe crab, Tachypleus tridentatus (instars 1-12, approximately 0.5-8 years old) across ontogeny was assessed in diversified nursery habitats along the northern Beibu Gulf, China, using carbon and nitrogen stable isotopes. Our results suggest that: (i) T. tridentatus are ecological generalists given the vast range of carbon isotopic values and trophic niche width estimates exhibited between multiple instar groups; (ii) juvenile T. tridentatus across most habitat types predominantly assimilated energy from a variety of basal production sources in the food web, but primarily depended on sedimentary organic matter and seagrass resource pools; (iii) ontogenetic shifts in juvenile dietary proportions were evident, with decreased reliance on sedimentary organic matter, coupled with increased reliance on benthic macroinvertebrate grazers, detritivores, and omnivores with age; and (iv) nearly all juvenile instars occupied similar trophic positions in the food web with slight shifts in trophic position present with increasing size. Our findings indicate that resource availability and ontogenetic diet shifts strongly influence horseshoe crab trophic dynamics, and age should be accounted when formulating habitat conservation measures based on resource use for Asian horseshoe crabs., (© 2020 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)