Fecal sterols are traditionally ascribed as important biomarkers for animal excrement, and have been widely used to identify the source of organic matter and to reconstruct paleoecological changes in Antarctic terrestrial, aquatic, and marine ecosystems. However, the in situ microbial hydrogenation of cholesterol to coprostanol could have significance as a proxy to infer paleoenvironmental studies in Antarctica, particularly in anoxic sediment. Here, we report that abundant coprostanol, which was traditionally deemed as a biomarker for human sewage contamination, was found in three anoxic sediment profiles (AC2, BI, and CH1), which were strongly influenced by animal excrement at North Victoria Land, western Ross Sea, Antarctica. Our results suggest that the high concentrations of coprostanol in these three sediment profiles were not due to animal excrement, since coprostanol is not present in penguin guano and is a minor component in seal excrement. Coprostanol/cholesterol and coprostanol/(coprostanol + cholestanol) ratios suggest that coprostanol in the sediment cores of AC2 and BI was primarily derived from bacterial hydrogenation of cholesterol introduced by penguin guano. Coprostanol in CH1 sediments is related to human sewage due to intensive research activities from 1968 to 2006 in this region. However, the low abundance of coprostanol and the ratios of coprostanol/cholesterol and coprostanol/(coprostanol + cholestanol) in a relatively oxidizing sediment core (IIL1) infer that coprostanol was likely contributed by seal settlement. Together with high-throughput sequencing of the 16S rRNA gene, the conversion of cholesterol to coprostanol by anaerobic bacteria (e.g., Eubacterium coprostanoligenes) could occur in anoxic aquatic systems. Our results suggest that the presence of coprostanol in Antarctic lacustrine sediment with anoxic conditions does not necessarily indicate seal activity and human waste as the microbial hydrogenation of cholesterol to coprostanol should also be considered.