The reliability of phytolith assemblage analysis for characterizing Mediterranean vegetation is investigated in this study. Phytolith assemblages are extracted from modern and buried Holocene soils from the middle Rhône valley (France). The relation between modern phytolith assemblages and the surrounding vegetation, as well as between fossil assemblages and contemporaneous vegetation, already reconstructed through other proxies, is discussed. We demonstrate that the main northwestern Mediterranean biomes are well distinguished by soil phytolith assemblage analysis. In particular, the density of pine and nonconiferous trees (densities expressed relatively to the grass cover) and the overall degree of opening of the vegetation appear well recorded by three phytolith indexes. North Mediterranean vegetation changes during the Holocene period, mainly tree line shifts, pine wood development and deforestation are poorly documented, due to the scarcity of proxy-preserving sites. Phytolith assemblage analysis of soils, buried soils, and sediments appears to be a promising technique to fill this gap.