Ancient (i.e., radiocarbon depleted) organic carbon (OC) is exported from ice sheet, glacier, and permafrost systems and may be buried, respired, or assimilated in downstream aquatic systems. Few studies have explored the potential use of this ancient OC in lake food webs. We combined natural abundance radiocarbon and stable carbon isotope data (Δ14C and δ13C) to study ancient OC utilization by zooplankton in six lakes covering a large climate gradient on the central and peripheral Tibetan Plateau. A depleted Δ14C signature of dissolved and particulate OC was found in the inflowing streams and lakes, ranging from – 49‰ to – 569‰, corresponding to radiocarbon ages between 403 yr and 6757 yr. The Δ14C values for zooplankton in the lakes ranged from – 45‰ to – 264‰, reflecting that zooplankton obtain 14C‐depleted signatures through assimilation of ancient OC and/or indirectly through consumption of phytoplankton or aquatic plant utilizing 14C‐depleted inorganic carbon. Moreover, ancient OC from inflowing streams contributed more to zooplankton diets in the temperate glacier area than in the cold glacier area. Assimilation of ancient OC by zooplankton in lakes is not only affected by drainage basin characteristics, such as the recharge coefficient of the lake, but also by the biogeochemical properties of OC. Use of ancient OC by zooplankton in high‐altitude lakes may constitute an important link between the contemporary aquatic food webs and the glaciated watersheds. Our findings have important implications for the contribution of ancient carbon to the modern lake food webs of high‐altitude and polar lakes.