A sub-population of giant Australian cuttlefish (Ascarosepion apama) in the upper Spencer Gulf of South Australia forms the only dense cuttlefish mating aggregation known globally. Though the aggregation forms on a confined, nearshore rocky reef, much of the lifespan of these cuttlefish occurs in deeper parts of the upper Spencer Gulf. The trophic niche of giant Australian cuttlefish through this phase of rapid growth is only partially understood, and may have unrecognised connections to species of commercial, recreational and ecological significance. We analysed δ13C and δ15N stable isotopes within the mantle tissue and beaks of giant Australian cuttlefish, plus the muscles of a range of small-bodied, potential prey species, finding that both juvenile and adult giant Australian cuttlefish have a broad diet that increases in trophic height throughout ontogeny. We also found potential trophic links to blue swimmer crabs (Portunus armatus) and New Holland cuttlefish (Ascarosepion novaehollandiae), suggesting that the fluctuating giant Australian cuttlefish population might have important effects on the upper Spencer Gulf food web.