The socio-cultural and economic developments that took place from the Neolithic to the Bronze Age are poorly understood, despite the fact that they were essential for the establishment of fully agro-pastoral economies in Europe. In this study, we aim to assess dietary changes in communities living in southern Italy during this period by examining stable carbon and nitrogen isotope ratios on human bone collagen. In particular, we investigated skeletal remains from seven sites in the southern Italian regions of Calabria (Grotta della Monaca, Grotta di Donna Marsilia and Grotta dell’Antenato), Basilicata (Murgia Timone, Grotta Funeraria and Toppo d’Aguzzo) and Apulia (Ipogeo dei Bronzi) to explore possible variations in diet between different geographic areas and periods. The results of the analysis on bone collagen extracts from 33 human and 12 faunal (sheep, dog, cattle and pigs) specimens attest that the diets of prehistoric southern Italians were mixed and based on the consumption of terrestrial resources, including generally moderate proportions of animal protein (e.g. meat and dairy products) and of C3 plants (e.g. cereals and legumes). Minor differences in the proportion of consumed meat are mostly dependent on the nature of regional environments, with individuals from Basilicata relying more on animal protein than those from Calabria and Apulia. Our study provides insights into the dietary habits of southern Italian populations during the prehistoric period that witnessed an increase both in agriculture and in pastoralism.