Anaerobic fungi are among the most active plant-degrading microbes in nature. Increased insight into the mechanisms and environmental cues that regulate fungal hydrolysis would better inform bioprocessing strategies to depolymerize lignocellulose. Here, we compare the response of three strains of anaerobic fungi (Piromyces finnis, Anaeromyces robustus, and Neocallimastix californiae) to catabolite regulation by simple carbohydrates. Anaerobic fungi exhibited high enzymatic activity against crystalline cellulose, which was repressed upon incubation with free sugars. Cellulolytic degradation was also inhibited when fungi were exposed to sugars they did not metabolize, suggesting a general mode of catabolite repression. RNA-Seq experiments in the presence of excess glucose confirmed repression of carbohydrate active enzymes during sugar uptake, and offer a path towards unmasking the function of co-regulated genes that could be involved in biomass degradation. Overall, these results suggest that sugar-rich hydrolysates tune the behavior of anaerobic fungi by dampening production of their biomass-degrading enzymes. (c) 2018 American Institute of Chemical Engineers