Opal, organic C and N contents, and organic δ13C and δ15N stable isotopes were analyzed on a sedimentary sequence spanning the past ~12,000 years from Dune Lake, a shallow groundwater-fed lake in the boreal forest region of central interior Alaska. The chronology, based on 210Pb and 15 AMS radiocarbon dates on terrestrial materials, indicates that 5 m of biogenic sediment accumulated following dune stabilization at ~10,000 cal yr BP. Previous studies on lake level and pollen were synthesized with these data to assess Holocene changes in hydrogeology and climate. Variability in groundwater level and flux has a strong control on lake level and are negatively correlated with the δ13C of organic matter produced within the lake. This relationship results from the input of groundwater with high pCO2 and with dissolved inorganic carbon depleted in 13C, which subsequently affects lake phytoplankton δ13C signatures. A rapid rise in lake level between ca. 11,000 and 9,500 cal yr BP coincided with stabilization of the dune field adjacent to the lake and expansion of vegetation around the lake, including white spruce. Organic δ13C values are relatively low and constant between about 10,000 and 6,000 cal yr BP, and then steadily increase until about 2,000 cal yr BP, suggesting a pattern of high followed by dropping lake levels generally consistent with results from core-transect studies. Higher-frequency fluctuations of 2–3 ‰ in δ13C are common, and suggest variability in aspects of the lake-carbon cycle over multi-decadal timescales. Recent trends in groundwater and lake level, river discharge, and precipitation suggest an important role for wintertime precipitation in the regional groundwater system. The unusual hydrology of this system relative to most other lakes studied for paleoclimate in this region provides a novel insight into different seasonal aspects of Alaskan paleoclimatology. The record is consistent with a general long-term decrease in wintertime precipitation, possibly accompanied by an increase in permafrost extent, over the Holocene.