The Neolithic Demographic Transition induced significant biological changes in human populations. Shift in dietary habits were introduced with the appearance of agro-pastoral subsistence economies. The central Balkan and southern part of Great Pannonian Plain represents one of the key areas for studying the Neolithization process as it is located at the crossroad between the Near East and central Europe. The rich anthropological collection of the southern part of Middle Danube Basin (Serbia; 6200-5500 BC) provides an opportunity to better understand the mechanisms of population’s adaptation to major ecological and socio-cultural changes. The main aim of this study is to understand the role of the Danube river and its influence on dietary strategies of first farmers and descendants of last hunter gatherers. This paper presents new insights on dietary adaptations along the Danube, comparing three lines of evidence: stable isotope data on bone collagen (C, N, S) providing information on the protein intake within the 10 last years of the individual life, buccal dental-microwear signatures to determine the dietary abrasiveness and the dental caries frequency related to carbohydrate intake in diet. Results suggest that the changes occurred in human palaeodietary behaviors following different patterns, in relation to both cultural adaptation and local environment along different parts of the Danube region. Our findings emphasize the importance of comparing different lines of evidence and to examine local behavioral adaptations in order to accurately understand the process of Neolithization and the impact of dietary and environmental factors.