ABSTRACT Oxygen-stratified lakes are typical for the boreal zone and also a major source of greenhouse gas emissions in the region. Due to shallow light penetration, restricting the growth of phototrophic organisms, and large allochthonous organic carbon inputs from the catchment area, the lake metabolism is expected to be dominated by heterotrophic organisms. In this study, we test this assumption and show that the potential for autotrophic carbon fixation and internal carbon cycling is high throughout the water column. Further, we show that during the summer stratification carbon fixation can exceed respiration in a boreal lake even below the euphotic zone. Metagenome-assembled genomes and 16S profiling of a vertical transect of the lake revealed multiple organisms in an oxygen-depleted compartment belonging to novel or poorly characterized phyla. Many of these organisms were chemolithotrophic, potentially deriving their energy from reactions related to sulfur, iron, and nitrogen transformations. The community, as well as the functions, was stratified along the redox gradient. The autotrophic potential in the lake metagenome below the oxygenic zone was high, pointing toward a need for revising our concepts of internal carbon cycling in boreal lakes. Further, the importance of chemolithoautotrophy for the internal carbon cycling suggests that many predicted climate change-associated fluctuations in the physical properties of the lake, such as altered mixing patterns, likely have consequences for the whole-lake metabolism even beyond the impact to the phototrophic community. IMPORTANCE Autotrophic organisms at the base of the food web are the only life form capable of turning inorganic carbon into the organic form, facilitating the survival of all other organisms. In certain environments, the autotrophic production is limited by environmental conditions and the food web is supported by external carbon inputs. One such environment is stratified boreal lakes, which are one of the biggest natural sources of greenhouse gas emissions in the boreal region. Thus, carbon cycling in these habitats is of utmost importance for the future climate. Here, we demonstrate a high potential for internal carbon cycling via phototrophic and novel chemolithotrophic organisms in the anoxic, poorly illuminated layers of a boreal lake. Our results significantly increase our knowledge on the microbial communities and their metabolic potential in oxygen-depleted freshwaters and help to understand and predict how climate change-induced alterations could impact the lake carbon dynamics.