The acidophilic and thermophilic pullulanases have many potential applications in the processes of starch liquefaction and saccharification. In this study, a gene encoding an amylopullulanase from Thermofilum pendens (TPApu) was heterologously expressed in Escherichia coli. Although TPApu possessed the same continuous GH57N_Apu domain and the succeeding α-helical region as other two amylopullulanases from Staphylothermus marinus (SMApu) and Caldivirga maquilingensis (CMApu), it only showed maximal amino acid identities of 25.7-28.7% with CMApu and SMApu. The purified TPApu appeared as a single band of SDS-PAGE with a molecular mass of 65.5 kDa and exhibited the maximal activity at pH 3.5 and 95-100°C. TPApu had the highest catalytic efficiency towards pullulan (kcat/km, 8.79 s-1 mL mg-1) and α-cyclodextrin (kcat/km, 0.36 s-1 mM-1). In the initial stages, the ring-opening reactions of γ-cyclodextrin, 6-O-glucosyl-β-cyclodextrin, 6-O-maltosyl-β-cyclodextrin and the debranching reactions of 6-O-maltooctaosyl-β-cyclodextrin were firstly catalyzed. In the subsequent reactions, a serial of maltooligosaccharides were produced. As the most acidophilic amylopullulanase among thermophilic pullulanases reported to date, TPApu preferred to debranch the DP6-12 side chains of amylopectin at pH 4.5 and 100°C.