Abstract An exo-inulinase gene from Kluyveromyces marxianus and an endo-inulinase gene from Aspergillus niger were cloned and expressed in Yarrowia lipolytica, respectively. Then, the purified recombinant exo-inulinase (rEXINU) and endo-inulinase (rENINU), either individual or combination with another, were used for hydrolysis of inulin and inulin in tuber meal of Jerusalem artichoke. After the analysis of the dynamic hydrolysis process, it was found that the rEXINU and the rENINU acted synergistically with each other, and the maximum degree of synergistic effect on the level of fructose formation (DSE Fru ) could be obtained with a molar ratio of the rEXINU to the rENINU at 1:1. Furthermore, the investigation of the hydrolysis product in the synergistic hydrolysis process showed that a wide range of oligosaccharides were first generated and then converted to fructose. Based on the enzyme kinetics, substrate specificity and product inhibition of the rEXINU and the rENINU, a synergism mechanism of the them for the inulin hydrolysis was proposed. During the process of the synergistic action, the rENINU preferentially degraded the inulin with high DP, but was inhibited by the produced oligosaccharides. Then the released oligosaccharides which were the optimum substrates of the rEXIUN were rapidly hydrolyzed by the enzyme to produce fructose and glucose. Meanwhile, this process relieved the inhibition of the produced oligosaccharides to the rENINU. Besides, in the synergistic hydrolysis of the inulin in tuber meal of Jerusalem artichoke, the depolymerization of the rENINU could result in the increase of the subtrate solubility to improve the hydrolysis efficiency. Therefore, this kind of the synergistic effect between the exo-inulinase and the endo-inulinase may provide a potential driving force in the industrial application of inulin and inulin-containing materials.