Carbon isotopes of soil organic matter (SOM) were used to evaluate and establish the chronology of the vegetation dynamics of an ecosystem presently composed of savannas surrounded by forests. The study was carried out on a 200-km transect along highway BR 319, on the border of Amazonas and Rondônia states, in southern Amazon, Brazil. Large ranges in δ13C values were observed in SOM collected from profiles in the savanna (−27 to −14‰) and forest regions (−26 to −19‰), reflecting changing distribution of 13C-depleted C3 forest and 13C-enriched C4 savanna vegetation in response to climate change. These results indicate that from about 17,000 to 9000 14C yr B.P., the study area was covered by forest vegetation. Between approximately 9000 and 3000 14C yr B.P., savanna vegetation expanded at the expense of the forest. Although the expansion of savanna did not occur with the same intensity along the study transect, this process was very clearly registered by 13C-enrichment in the SOM. Since 3000 14C yr B.P., the carbon isotope data suggest that forested regions have expanded. This study adds to the mounting evidence that extensive forested areas existed in the Amazon during the last glaciation and that savanna vegetation expanded in response to warm and dry conditions during the early to middle Holocene.