The Middle Neolithic of the Northwestern Mediterranean area (∼4500–3500 BC cal) is characterized by the development of food production techniques as well as by increasing social complexity. These characteristics could have had an impact on human dietary patterns. To evaluate human dietary practices and lifeways of the Middle Neolithic populations from the South of France, stable carbon and nitrogen isotope analysis was carried out on 57 human and 53 faunal bones from seven archaeological sites located in the Languedoc and Garonne regions between 20 and 100 km from the Mediterranean Sea, respectively. Results show regional differences in carbon isotope values. Animal and human bones from the Languedoc region are significantly enriched in 13C relative to the Garonne. Conversely, human and dog bones from the Garonne region are significantly enriched in 15N compared to human and dog bones from the Languedoc region. These results highlight the importance of the local ecosystem in human and animal diet as well as a regional differentiation of palaeodietary behavior, which probably relates to economic and social factors. The comparison of stable isotope data with archaeological and biological evidence does not show any significant intra- or interpopulation differences. However, the presence of human outliers suggests that migration probably occurred, perhaps in relation to the trade of animals and/or materials. This study also highlights the importance of investigating local animal stable isotope values for the interpretation of human palaeodiet. Am J Phys Anthropol 2010. © 2009 Wiley-Liss, Inc.