The paper aims to point out the subsistence in Eneolithic Central Italian communities by Stable Isotope Analysis. This period marked a tipping point in the food strategies because it was characterized by economic changes and several technological improvements leading to enhance land exploitation and livestock breeding. Carbon and nitrogen stable isotope analysis has been used to analyze the food consumption of 54 people belonging to 5 Eneolithic communities scattered throughout Central Italy, where no data have yet been published. The estimation of the main protein intake has been achieved in order to quantify the differences among these communities. The results are consistent with a diet mainly based on terrestrial resources, with no exclusive marine sources consumption, although their occasional usage cannot be ruled out, especially for selected funerary contexts. The data suggest an overall subsistence based on greater local resource procurement, supported by regional productivity maximization. A roughly homogeneous landscape could be outlined in Tuscany and Marche communities witnessing a shared diet preference that could be modified by local preferences. The fully developed trade routes between the two sides of the Apennines could address the overall dietary homogeneity of the studied communities, especially between Fontenoce di Recanati and the southern Tuscan human groups such as Grotta del Fontino and Buca di Spaccasasso, with lesser influence for Le Lellere and Podere Cucule that seem to suggest a more locally based subsistence, even though the funerary affinities do not match this overall diet homogeneity.