The thermophilic filamentous ascomycete Chaetomium thermophilum produces functionally diverse hemicellulases when grown on hemicellulose as carbon source. Acetyl xylan esterase (EC 3.1.1.72) is an important accessory enzyme in hemicellulose biodegradation. Although the genome of C. thermophilum has been sequenced, its carbohydrate esterases are not annotated yet. We applied peptide pattern recognition (PPR) tool for sequence analysis of the C. thermophilum genome, and 11 carbohydrate esterase genes were discovered. Furthermore, we cloned and heterologously expressed two putative acetyl xylan esterase genes, CtAxeA and CtAxeB, in Pichia pastoris. The recombinant proteins, rCtAxeA and rCtAxeB, released acetic acids from p-nitrophenyl acetate and water-insoluble wheat arabinoxylan. These results indicate that CtAxeA and CtAxeB are true acetyl xylan esterases. For both recombinant esterases, over 93 % of the initial activity was retained after 24 h of incubation at temperatures up to 60 °C, and over 90 % of the initial activity was retained after 24 h of incubation in different buffers from pH 4.0 to 9.0 at 4 and 50 °C. The overall xylose yield from wheat arabinoxylan hydrolysis was 8 % with xylanase treatment and increased to 34 % when xylanase was combined with rCtAxeA and rCtAxeB. In sum, the present study first report the biochemical characterization of two acetyl xylan esterases from C. thermophilum, which are efficient in hydrolyzing hemicellulose with potential application in biomass bioconversion to high value chemicals or biofuels.