The molecular distributions and isotopic ratios (δ13C and δD) of n-alkanes in sediments deposited during the last 35 cal kyr BP in Saci Lake, southeastern Amazonia, have been measured and interpreted for their paleoclimate information. A terrestrial origin for the odd carbon-numbered long-chain (>C27) n-alkanes has been inferred from molecular distributions. A shift in the n-alkane δ13C values across the Pleistocene-Holocene boundary coincides with a climate-induced floral change previously deduced from palynological analyses. Dry conditions during the late Pleistocene and mid-Holocene were confirmed by several n-alkane indices used to estimate the availability of moisture and by larger δD values. High ACL values indicate relatively dry conditions from 29 to 18.2 cal kyr BP and again from 8.0 and 5.0 cal kyr BP with a decrease in precipitation and an increase of grasses. Changes in the n-alkane C-29 isotopic compositions (δD and δ13C) were observed during the Pleistocene-Holocene transition, coinciding with a floristic change induced by climatic conditions that is also observed in palynological results from the same lake. The n-alkane C-29 δ13C values remain between −33.2‰ and − 35.7‰, which is representative for n-alkanes derived from C3 vegetation. These lines of evidence suggest a continuous gallery forest cover in the Saci Lake catchment for the last ~35 cal kyr BP in contrasted with the observed palynological changes which represent the dynamics of the vegetation on the upland areas. The n-alkane C-29 δD values are less negative in the Pleistocene and more negative during the Holocene in the lake record. These characteristics suggest more humid conditions during the Holocene than in the Pleistocene, probably associated with the South American Monsoon System (SAMS). However, an increase of approximately 10‰ occurs during the late Holocene, which coincides with the forest expansion around 5 cal kyr BP that is recorded in palynological data of Saci Lake and other records of the Amazon Forest. This change suggests that the D/H ratio is also influenced by the increase of the plant biomass, which would provide an enrichment of D in the isotopic signal as a function of regional moisture recycling caused by evapotranspiration.