Dissolved organic carbon (DOC) derived from thermokarst lakes is usually considered to be prone to microbial degradation and releases substantial carbon dioxide to the atmosphere, potentially enhancing the positive permafrost carbon (C)-climate feedback. In contrast to this long-term standing view, here we show that dark C fixation exceeds DOC degradation in ~1/3 of the investigated thermokarst lakes on the Tibetan Plateau, based on the combination of large-scale water and sediment sampling across seasons and years, biodegradable DOC experiments and 14C-labeling bicarbonate (NaH14CO3) assimilation incubation experiment. By employing qPCR, amplicon sequencing and metagenomic analyses, we find that microbial C fixation is mainly driven by nitrifying microorganisms via the Calvin-Benson-Bassham cycle carried out by the cbbL gene (encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase). These findings demonstrate that chemoautotrophic C fixation predominates in part of thermokarst lakes, which could partly offset C emissions upon permafrost thaw and thus weaken the positive permafrost C-climate feedback.