In Southern France as in other parts of Europe, significant changes occurred in settlement patterns between the end of Antiquity and the beginning of the Middle Ages. Small communities gathered to form, by the tenth century, villages organized around a church. This development was the result of a new social and agrarian organization. Its impact on lifestyles and, more precisely, on diet is still poorly understood. The analysis of carbon and nitrogen isotopes in bone collagen from the inhabitants of the well-preserved medieval rural site Missignac-Saint Gilles le Vieux (fifth to thirteenth centuries, Gard, France) provides insight into their dietary practices and enables a discussion about its transformation over time. A sample of 152 adult individuals dated from 675 to 1175 AD (75 females, 77 males) and 75 specimens from 16 non-human species were analyzed. Results show the exploitation of freshwater, marine, and terrestrial ecosystems as well as various breeding practices specific to each species. The use of both C4 and halophyte plants for feeding domestic animals was also observed. Concerning human dietary practices, a change seemed to occur at the beginning of the tenth century with an increase of δ15N values and a decrease of δ13C values. This corresponds to the introduction of a significant amount of freshwater resources into the diet and could be related to the evolution of the Catholic doctrine. A concomitant diversification of access to individual food resources was also observed, probably linked to the increased diversity of practice inside a population otherwise perceived as one community.