Possible changes in diet and trophic levels in relation to size of Mediterranean bluefin tuna, Thunnus thynnus, were investigated using labelled carbon (δ13C) and nitrogen (δ15N) stable isotopes. Samples were obtained from two locations in the southern Tyrrhenian Sea (Western Mediterranean Sea) in May and October 2004. The δ13C and δ15N analyses revealed at least three significant isotopic groups [small juveniles (0.7–2.2 kg), sub-adults (15–50 kg) and adults (70 to 225 kg)]. δ13C was negatively dependent on weight, while δ15N was positively dependent on weight [TW = 8.2 (±0.16) + 0.03 (± 0.0) *δ15N (n = 49; r = 0.91; P < 0.001)]. Different prey contribution to the diet was highlighted for each class. The diet of juveniles comprised zooplankton, small pelagic fish and some coastal fish; sub-adults relied on medium pelagic fish, shrimps and cephalopods, and adults relied mainly on cephalopods and larger fish. The trophic level (TL) of tunas belonging to each size class was closely correlated to weight, starting from ca 3.0 TL for Group I and reaching 4.4–4.8 TL for the giants. Bluefin tuna, from small juveniles to giants, showed a shift in feeding preferences due to different use of habitats and food items as a function of the life stage.