This article presents a new comprehensive assessment of the Holocene hydrological variability of Lake Ladoga, northwest Russia. The reconstruction is based on oxygen isotopes of lacustrine diatom silica (δ18Odiatom) preserved in sediment core Co 1309, and is complemented by a diatom assemblage analysis and a survey of modern isotope hydrology. The data indicate that Lake Ladoga has existed as a freshwater reservoir since at least 10.8 cal. ka BP. The δ18Odiatom values range from +29.8 to +35.0‰, and relatively higher δ18Odiatom values around +34.7‰ between c. 7.1 and 5.7 cal. ka BP are considered to reflect the Holocene Thermal Maximum. A continuous depletion in δ18Odiatom since c. 6.1 cal. ka BP accelerates after c. 4 cal. ka BP, indicating Middle to Late Holocene cooling that culminates during the interval 0.8–0.2 cal. ka BP, corresponding to the Little Ice Age. Lake‐level rises result in lower δ18Odiatom values, whereas lower lake levels cause higher δ18Odiatom values. The diatom isotope record gives an indication for a rather early opening of the Neva River outflow at c. 4.4–4.0 cal. ka BP. Generally, overall high δ18Odiatom values around +33.5‰ characterize a persistent evaporative lake system throughout the Holocene. As the Lake Ladoga δ18Odiatom record is roughly in line with the 60°N summer insolation, a linkage to broader‐scale climate change is likely.